ameliorative effects of infantile feire kechuan oral

Research ArticleAmeliorative Effects of Infantile Feire KechuanOral Solution on Mycoplasma Pneumoniae Pneumonia inInfant Mouse and Rat Models

Shanshan Guo1 Lei Bao 1 Tiange Qu 2 Xin Mao 1 Yingjie Gao1

Yingli Xu 1 and Xiaolan Cui 1

1 Institute of Chinese Materia Medica China Academy of Chinese Medical Sciences No 4 Yinghua East RoadChaoyang District Beijing 100029 China2Beijing University of Chinese Medicine No 11 Bei San Huan Dong Lu Chaoyang District Beijing 100029 China

Correspondence should be addressed to Xiaolan Cui cuixiaolan2812126com

Received 31 March 2018 Revised 8 August 2018 Accepted 16 August 2018 Published 17 September 2018

Academic Editor Francisco Solano

Copyright copy 2018 Shanshan Guo et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Mycoplasma pneumoniae (MP) infection is a major pathogen of community-acquired pneumonia (CAP) in children worldwideInfantile Feire Kechuan Oral Solution (IFKOS) has been used for the treatment of MP pneumonia clinically in China for manyyears The present study was designed to investigate the therapeutic effect of IFKOS on MP pneumonia and explore the potentialmechanismof the actionsThe infant BALBcmouse andWistar ratmodels ofMP infectionwere successfully established to confirmthe therapeutic effects of IFKOS followed by assays for related cytokines and investigations of the IgM response involvedThe resultsshowed that IFKOS exhibited an inhibitory effect on pulmonary index (PI) and effectively reduced the degree of lesions in the lungsThe lethal rate of mice was significantly decreased while survival time of mice was dramatically increased by IFKOS treatment incomparison to infection control respectively IFKOS treatment (40 20 and 10mlkg) significantly decreased the level ofMP-IgM ina dose-dependent manner whereas IFKOS showed no obvious inhibitory effect on the increase of relative expression of MP-DNAIn addition the elevated IL-2 and TNF-120572 levels were significantly reduced and the decreased IL-6 level was significantly enhancedby IFKOS treatment Our study demonstrates that IFKOS has inhibitory effect on MP infection in infant mouse and rat models ofMP pneumonia and protective effect from lethal MP challenge in infant murine model These anti-MP effects might be related tosuppression of the IgM response and a reversal the imbalance of Th1Th2 cytokines induced by MP infection

1 Introduction

Mycoplasma pneumoniae (MP) is one of the most commonpathogens of community- acquired pneumonia (CAP) inyoung adults and children and is a significant worldwidecause of morbidity and mortality in both adult and pediatricpopulations [1] MP accounts for 3ndash10 of pneumonia casesand 10-30 of all CAP cases [2 3] especially in east Chinaand MP infection has been detected in approximately 50 ofthe CAP population [4] Although MP pneumonia in mostpatients is generally mild or moderate with only 3-13of infected persons developing pneumonia in recent yearsincreasing numbers of severe pneumonia in children fromMP infection have been reported including many fatal cases

[5 6] MP not only causes upper and lower respiratory tractinfections but also leads to extrapulmonary complicationswhich affect almost every organ [7]

It has been demonstrated that an excessive host immuneresponse against pathogens may play a pivotal role in refrac-toryMycoplasma pneumoniae pneumonia (RMPP) in school-aged children including overexpression of cytokines suchas interleukin-1szlig (IL-1 szlig) interleukin-6 (IL-6) interleukin-10 (IL-10) interferon gamma (IFN-120574) tumor necrosis factoralpha (TNF-120572) and lactate dehydrogenase (LDH) [8ndash10]The severity of Mycoplasma pneumoniae lung disease maybe related to the degree to which the host immune responsereacts to the infection based on evidence from clinicalresearch and from studies in vitro and in vivo [11] It was

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 8139040 10 pageshttpsdoiorg10115520188139040

2 Evidence-Based Complementary and Alternative Medicine

found that IL-8 IL-10 and IL-18 appeared to modulatepulmonary lesions inMP pneumonia in children of a medianage of 5 years [12] In addition the production of IL-2 IL-4IL-10 and IFN-120574 by splenic lymphocytes has been detectedin significant high levels in a gnotobiotic mice model ofMycoplasma pneumoniae pneumonia and IL-1szlig and IL-6were upregulated in a novel mouse model immunized withMP extracts plus alum [13 14]

Macrolides have been recommended as the first-linetherapy in adults whereas azithromycin (AZM) as thefirst-line drug in for pediatric cases [15 16] Howevermacrolide-resistant MP pneumoniae (MRMP) pneumoniahave emerged and became widespread in East Asia since2000 In the last decade macrolide resistance rates in MRMPpneumoniae among children have substantially increasedworldwide [17 18] especially inChina and Japan where it hasreached up to 90 [19 20] Therefore it is extremely urgentto develop new drugs as safe and effective alternative agentsfor the treatment of MP pneumonia

Infantile Feire Kechuan Oral Solution (IFKOS) is atraditional Chinese medicine that is mainly used to treatrespiratory tract infections such as influenza and pneumoniain children IFKOS is composed of the following Chinesemedicines Lonicerae japonicae Flos Ephedrae Herba Gly-cyrrhizae Radix et Rhizoma Forsythiae Fructus Scutellar-iae Radix and Armeniacae Semen amarum Studies havereported that active constituents of IFKOS such as chloro-genic acid and forsythiaside may have pharmacological func-tions of wind dispelling heat-clearing detoxifying and painrelieving [21 22] In China IFKOS has been listed as a pro-prietary Chinese medicine to combat pediatric respiratoryinfection Many clinical studies have reported the curativeeffects of IFKOS against MP pneumonia and the cure ratewas up to 9070 when infantile MP pneumonia was treatedwith IFKOS combined with azithromycin [23ndash25] Howeverfew studies have investigated the curative and underlyingmechanisms of the actions involved in experimental models

In the present study the therapeutic effect and mech-anisms of IFKOS on MP pneumonia were assessed usinginfancy BALBc mouse and Wistar rat models Quantitativereal-time polymerase chain reaction and an enzyme-linkedimmunosorbent assay (ELISA) were used to evaluate thetherapeutic effect of IFKOS on the expression levels of MP-DNA MP-IgM IL-2 IL-6 and TNF-120572 in response to MPpneumonia In addition pulmonary injury in mice and ratswas detected by pathological examination under microscopeand the mortality rate and survival time in a BALBc micemodel were observed

2 Materials and Methods

21 Test Drugs All investigations were performed with asingle batch (No 201610001) of IFKOS kindly provided by themanufacturer Kuihua Pharmaceutical (Heilongjiang China)The doses of IFKOS were 40 20 and 10mlkgday for infantmice and 20 10 and 5mlkgday for infant rats All the dosesfor mice and rats were equivalent to two one and half timesthe clinical dose of 6-year-old children (2mlkg) accordingto the ratio of body surface area of human and mice or rats

respectively Azithromycin (batch No 063160821) was pro-duced by Ouyi Pharma China Shijiazhuang PharmaceuticalGroup served as a reference drug The therapeutic dose ofazithromycin is 5 mgkg daily (15-25kg children) and thedoses for mice and rats were 50 and 25mgkgday accordingto the ratio of body surface area of human and mice or ratsrespectively

22 Animals Male and female BALBc mice (13-15g) andWistar rats (90-110g) were purchased from Vital River Lab-oratory Animal Technology Co Ltd in Beijing China(Certificate No SCXK 2016-0011) and maintained in anenvironmentally controlled breeding room (temperature 20plusmn 2∘C humidity 50 plusmn 10) The study was carried out inaccordance with the rules of European Community Guide-lines for care and use of animals The study was approvedby Ethics Committee at Institute of Chinese Materia MedicaChina Academy of Chinese Medical Sciences

23 MP Culture MP FH strain (ATCC15531 AmericanType Culture Collection Rockville MD USA) was culturedin modified mycoplasma medium (Oxoid UK) containingPPLO broth horse serum and 25 yeast extract to whichwas added penicillin G (200 Uml) thallium acetate (0025) glucose (1 ) and phenol red (0002 ) pH 78 MP wascultured at 37∘Cunder 5 CO

2for 7 daysThe concentration

of MP was determined as 108 color change units (CCU)mlaccording to the color-change-unit method [26]

24 Mouse Model of MP Pneumonia BALBc mice wererandomly divided into six groups with 10 mice per group(half male and half female) The groups were assignedas normal control group infection control group andfour treatment groups (three IFKOS treatment groups andone positive drug-azithromycin treatment group) All miceexcept those in the normal control group were repeatedlyinfected intranasally with 50120583L of the MP solution at aconcentration of 108CCUml for three times once a day Inthe treatment groups the inoculated mice were successivelygiven IFKOS orally at a dose of 40mlkg 20mlkg or 10mlkgor treated with azithromycin at a dose of 50mgkg daily for3 days beginning from the day of infection Each mouse ofthe control group was given an equal volume of distilledwater On day 4 blood of all the mice was collected andthe serum was separated to determine the level of MP-IgM Then the mice were sacrificed and the body weight(BW) and lung weight (LW) were measured to calculatethe pulmonary index (PI) and inhibitory rate of pulmonaryindices (IRPI)PI=[LW(g)BW(g)]times100IRPI=[(LW of infec-tion control group-LW of treatment group)(LW of infectioncontrol group-LW of normal control group)]times100 The lungtissues were extracted and subjected to qRT-PCR detectionand histological examination

25 Rat Model of MP Pneumonia Wistar rats were ran-domly divided into six groups with 10 rats per group (halfmale and half female) as described above All rats exceptthose in the normal control group were repeatedly infectedintranasally with 150120583L of the MP solution at a concentration

Evidence-Based Complementary and Alternative Medicine 3

of 108CCUml for four times once a day In the treatmentgroups the inoculated rats were successively given IFOKSorally at a dose of 20mlkg 10mlkg or 5mlkg or treatedwith azithromycin at a dose of 25mgkg daily for 4 daysbeginning from the day of infection Each rat of the controlgroup was given an equal volume of distilled water On day 5all the rats were sacrificed and the lung tissues were extractedand subjected to ELISA assay and histological examination

26 Mouse Model of Death due To MP Infection A totalof 100 BALBc mice were randomly distributed into fivegroups with 20 mice per group including infection controlgroup azithromycin treatment group and three IFKOStreatment groups All the mice were repeatedly infectedintranasally with 60120583L of the MP solution at a concentrationof 108CCUml for five times once a day In the treatmentgroups the inoculated mice were successively given IFKOSorally at a dose of 40mlkg 20mlkg or 10mlkg or treatedwith azithromycin at a dose of 50mgkg daily for 5 days atthe beginning of the infection day Each mouse of the controlgroup was given an equal volume of distilled water The dayafter the last infection the number of deaths in each groupwas recorded for 14 consecutive days

27 Quantitative Real-Time PCR Detection of MP-DNADNA samples were extracted from the left lobes of allmice lungs using the DNeasy Blood amp Tissue kit (Qia-gen Hilden Germany) according to the manufacturerrsquosprotocol The presence of MP-DNA was diagnosed usingMP Real-Time PCR kit (Zhi-Jiang Co Ltd ShanghaiChina) with the following PCR amplification system DNAtemplate (4120583L) MP PCR reaction mix (35120583L) and Taqenzyme (04120583L) In the real-time PCR SYBR green wasused as fluorescence reporter specific forward primer 51015840-CAGAAACACACACAGCAGCTATT-31015840 and reverse primer51015840-CACGTTGATCCGCAAAGGAAGT-31015840 The PCR reac-tion was carried out using a quantitative PCR instrument(Bio-Rad CFX96 USA) under the following conditions 37∘Cfor 2 min 94∘C for 2 min followed by 40 cycles of 93∘C for 15s and 60∘C for 1 min

28 ELISA Assay for MP-IgM IL-2 IL-6 and TNF-120572 Thelevel of MP-IgM in mice serum was determined by anenzyme-linked immunosorbent assay(ELISA) kit (96T Meil-ian Co Ltd Shanghai China) according to the manufac-turerrsquos instructions and the levels of IL-2 IL-6 and TNF-120572 inrat lung tissues weremeasured using ELISA kits (96TMeilianCo Ltd Shanghai China)

29 Histological Examination The right lobes of the lungsin mice or rats were fixed in 10 paraformaldehyde thentrimmed and embedded in paraffin and cut into slices(3mm) for hematoxylin and eosin (HampE) staining Morpho-metric analysis was performed using an optical microscope(Leica DMLB-HC Germany) to evaluate the histopathologi-cal changes of the lungs

The following pulmonary infection score system wasapplied for grading the pulmonary lesion

Table 1 Inhibitory effect of IFKOS on PI in mice MP pneumoniamodel

Group PI IRPI()Normal control 068plusmn012 -Infection control 156plusmn032lowastlowast -azithromycin 128plusmn028 3261IFKOS(40mlkg) 108plusmn012 5493IFKOS(20mlkg) 129plusmn022 3157IFKOS(10mlkg) 123plusmn014 3778Each group represented the value of PI and IRPI PI was expressed as meanplusmn SEM (n = 10) lowastlowastp lt 001 compared to normal control group plt001plt005 compared to infection control group

ldquo0rdquo pulmonary alveoli showing no fusion no alveo-lar interstitial infiltration of inflammation cells noinflammation cells around the bronchi and no shed-ding in the lumen

ldquo1rdquo pulmonary alveoli showing no fusion but with local-ized pulmonary interstitial infiltration of lympho-cytes and macrophagocytes inflammation cells limi-tation inflammation cells around the bronchi and noshedding in the lumen

ldquo2rdquo pulmonary alveoli showing no obvious enlargementmild pulmonary interstitial infiltration of lympho-cytes and macrophagocytes inflammation cells mildinflammation cells around the bronchi and no shed-ding in the lumen

ldquo3rdquo obvious pulmonary interstitial infiltration of lympho-cytes and macrophagocytes inflammation cells pul-monary interval widened significant inflammationcells around the bronchi and no obvious shedding inthe lumen

ldquo4rdquo a large area of diffuse pulmonary interstitial infiltra-tion of lymphocytes andmacrophagocytes inflamma-tion cells pulmonary interval widened a large area ofdiffuse inflammation cells around the bronchi and noobvious shedding in the lumen

210 Statistical Analysis The GraphPad Prism 60 softwaresystem was employed for statistical analysis

All data are presented as meanplusmnSEM (standard error ofthe mean) A 1205942 test was used to statistically analyze thereduction in mortality other results were analyzed by one-way analysis of variance (ANOVA) and significant differ-ences were determined by the Bonferroni Test P valuesle005were taken as statistically significant

3 Results

31 IFKOS Inhibits MP-Induced High Pulmonary Index (PI) inMice As shown in Table 1 mice from the infection controlgroup presented an increase in PI (156plusmn032) compared tothe normal control group (068plusmn012) After 3-day treatmentthere was an obvious decrease of PI in all three IFKOS groupscompared with the infection control group In additionIFKOS exhibited significant PI inhibitory activity and the


5

45

4

35

3

25

2

15

05

0

1

normal group infection group azithromycin IFKOS(40mlkg) IFKOS(20mlkg) IFKOS(10mlkg)

group

rela

tive e

xpre

ssio

n of

MP-

DN

D(lo

g va

lue o

f cop

oes)

(a)


group

180

160

140

120

100

80

60

40

20

0leve

l of M

P-Ig

M (n

gm

l)

(b)

Figure 1 Anti-Mycoplasma pneumoniae activity of IFKOS in infancy BALBc mice model (a) The relative expression of MP-DNA wasmeasured by qRT-PCR Relative log values of MP copies were expressed as the mean plusmn SEM (n=6) lowastlowastp lt 001 compared to normal controlgroup p lt 005 compared to infection control group (b)The level of MP-IgM was measured by ELISA assay Each value was represented asmeanplusmn SEM (n=10) lowastp lt 005 compared to normal control group p lt 001 compared to infection control group

inhibitory rate of pulmonary indices (IRPI) were 54933157 and 3778 at the doses of 40 20 and 10mlkgrespectively

32 Anti-Mycoplasma pneumoniae Activity of IFKOS In thecurrent study the anti-MP activity of IFKOS was measuredusingMP-DNAandMP-IgM in lungs or serumTodeterminewhether the replication of MP was inhibited by IFKOSdirectly in mice we further examined the relative expressionof MP-DNA In addition the level of MP-IgM in serum wasdetected to discover the effect of IFKOS on immune responseinduced by MP infection As shown in Figures 1(a) and 1(b)the relative expression of MP-DNA increased dramaticallyin the infection control group whereas IFKOS showed noobvious inhibitory effect on the increase of relative expressionofMP-DNAwhich demonstrates thatMP replication inmicewas not inhibited directly by IFKOS treatment The levelof MP-IgM in the infection group increased significantlycompared to the normal control group IFKOS treatment (4020 and 10mlkg) significantly decreased the level of MP-IgMin a dose-dependent manner which indicated that elevationof MP-specific IgM was significantly depressed by IFKOStreatment in a dose-dependent manner

33 Inhibitory Effect of IFKOS on MP Pneumonia in Mice andRats After MP pneumonia mice were treated with IFKOS(40 20 and 10mlkg) for 3 days the lung tissues wereisolated for HampE staining As shown in Figures 2(a)ndash2(f)in the normal control group the lung tissues were whollyunaffected and there were no lesions in pulmonary alveolusand alveolar interstitium In the infection group a large areaof diffuse pulmonary interstitial infiltration of lymphocytesand macrophagocytes inflammation cells was observed aswell as widened pulmonary interval In the IFKOS treatmentgroup the pulmonary interstitial infiltration of inflammationcells was decreased As shown in Table 2 the score of lesionsin the lungs was significantly reduced in mice treated withIFKOS at the dose of 10mlkg

As shown in Figures 3(a)ndash3(f) in the normal controlgroup there were no inflammation cells around the bronchiand no shedding in the lumen In the infection groupsignificant inflammation cells were observed around the

Table 2 Effect of IFKOS on pulmonary lesion of mice or ratsinfected with MP

Group Number of animals mice ratslung bronchus lung

Normal control 10 0 0 0Infection control 10 29lowastlowast 24lowastlowast 21lowastlowast

azithromycin 10 22 21 13

IFKOS(high dose) 10 23 25 13

IFKOS(middle dose) 10 24 2 13

IFKOS(low dose) 10 21 24 14Each group represented the mean score of pulmonary lesion of mice or ratsinfected with MP lowastlowastp lt 001 compared to normal control group p lt001p lt005 compared to infection control group

bronchi but without any obvious shedding in the lumenHowever as shown in Table 2 no significant effect of IFKOStreatment on bronchial lesion was seen

After MP pneumonia rats were treated with IFKOS (2010 and 5mlkg) for 4 days histopathological examination wasconducted to reveal significant pathological changes in lungsof rats As shown in Figures 6(a)ndash6(f) in the normal controlgroup lung tissue retained its basic structure In the infec-tion group pulmonary interstitial infiltration of lympho-cytes and macrophagocytes inflammation cells was notedin alveolar interstitium and the pulmonary interval waswidened In the IFKOS treatment group the pulmonaryinterstitial infiltration of inflammation cells was decreasedAs shown in Table 2 the score of lesions in the lungs wassignificantly reduced when treated with IFKOS at the dosesof 20 and 10mlkg

34 IFKOS Treatment Protects Mice from Lethal MP Chal-lenge To evaluate the protective efficacy of IFKOS againstlethal MP challenge BALBc mice were subjected to sequen-tial infection for five times As shown in Figure 4(a) in the 14days after the last infection all the mice in the infection groupdied The lethal rate was significantly decreased to 55 byIFKOS treatment at a dose of 40mlkg compared to infectioncontrol Furthermore IFKOS treatment protected 920 520and 120 mice (45 25 5) from death at the doses of


(a) (b) (c)

(d) (e) (f)

Figure 2 Pathological changes in lung tissues of BALBc mice After treatment with IFKOS for 3 days the right lobes of the lungs in micewere fixed in 10 paraformaldehyde embedded in paraffin and cut into slices for HampE staining Morphometric analysis was performedusing an optical microscope (a) Normal control group (b) infected control group (c) azithromycin treatment group (d) IFKOS (40mlkg)treatment group (e) IFKOS (20mlkg) treatment group and (f) IFKOS (10mlkg) treatment group

(a) (b) (c)

(d) (e) (f)

Figure 3 Pathological changes in bronchi of BALBc mice (a) Normal control group (b) infected control group (c) azithromycin treatmentgroup (d) IFKOS (40mlkg) treatment group (e) IFKOS (20mlkg) treatment group and (f) IFKOS (10mlkg) treatment group


100

80

60

40

20

00 12108642

infection controlazithromycinIFKOS(40mlkg)IFKOS(20mlkg)IFKOS(10mlkg)

day post infection14

Perc

ent s

urvi

val

(a)

0

12

10

8

6

4

2 surv

ival

tim

e (da

y)

infection group azithromycin IFKOS(40mlkg) IFKOS(20mlkg) IFKOS(10mlkg)group

(b)

Figure 4 Protective effect of IFKOSonBALBcmice from lethalMP challengeThemicewere repeatedly infectedwith 60120583LMP (108CCUml) for five times once a day After treatment with IFKOS for 5 days the number of deaths in each group was recorded for 14 consecutive days(n=20) (a) Survival rate of MP infected mice treated with IFKOS (40 20 and 10mlkg) in the 14 consecutive days (b) IFKOS increasedsurvival time (day) of mice in a dose-dependent manner plt001 compared to infection control group

250

200

150

100

50

0

leve

l of T

NF-

(n

gm

l)


group

(a)

300

250

200

150

100

50

0

leve

l of I

L-2

(ng

ml)


group

(b)

600

500

400

300

200

100

0

leve

l of I

L-6

(ng

ml)


group

(c)

Figure 5 Effect of IFKOS on levels of cytokines in lung tissues of infancy rats infected by MP Values were expressed as the mean plusmn SEM(n=10) lowastlowastp lt 001 lowastp lt 005 compared to normal control group p lt 001 p lt 005 compared to infection control group (a) The level ofTNF-120572 (b) The level of IL-2 (c) The level of IL-6

40 20 and 10mlkg respectively As shown in Figure 4(b)IFKOS treatment (40 and 20mlkg) dramatically increasedsurvival time of mice to 795 d and 550 d in comparison tothe infection control respectively

35 Effect of IFKOS on Cytokines Secretion Induced by MPMany cytokines play a crucial role in inflammatory responsestriggered by MP To determine whether IFKOS exhibitedanti-MP pneumonia activity by inhibiting cytokine secretionIL-2 IL-6 and TNF-120572 in lung tissues were determined

by ELISA As shown in Figure 5 the levels of IL-2 andTNF-120572 were significantly increased while the level of IL-6was obviously decreased in the infection group comparedwith the normal control group However IFKOS treatment(2010 5mlkg) significantly decreased the level of IL-2 ina dose-dependent manner The elevated TNF-120572 level wassignificantly decreased by IFKOS at the dose of 20mlkg Inaddition a notable increase of IL-6 level was observed inthe IFKOS (10 5mlkg) groups in comparison with the nor-mal control group These results demonstrate that cytokine


secretion induced by MP infection was significantly reversedby IFKOS treatment

4 Discussion

Although MP pneumonia is generally benign and self-limiting several studies have indicated that MP can progressinto severe life-threating diseases such as refractory MPpneumonia acute respiratory distress syndrome necrotiz-ing pneumonitis and fulminant pneumonia in school-agedchildren from 5 to 15 years [27 28] However as overallmacrolides have been extensively used in children the occur-rence of MRMP has led tomore severe cases requiring hospi-talization worldwide [29]Themain mechanism of resistanceis mutations at the sites 2063 2064 2067 and 2617 inmutations in domainVof 23S rRNA [30]MRMPpneumoniacauses more severe clinical outcomes in children including alonger duration of fever cough and hospital stay in additionto radiologic progression [15] Thus there is a high unmetmedical need for MP pneumonia treatment for children onewhich is effective for severe and fulminant disease

Research on MP pneumonia usually focuses on detectingthe physiological and biochemical characteristics of thepatients and clinical treatment experience Fewer studieshave reported on the effects of traditional Chinese medicineon experimental animal models of MP infection IFKOShas been used to treat MP pneumonia for many years inChina and clinical results have showed that this drug couldeffectively alleviate its symptoms and could also reduce thefever cough radiologic progression disappearance time andthe average hospitalization time in children [31 32] Thepresent study was designed to demonstrate the anti-MPactivity of IFKOS and to investigate the pharmacologicalmechanism of anti-MP pneumonia action

In this study we successfully established the infant mouseand rat models of MP infection according to reports in theliterature [33 34] The therapeutic effects were confirmed inthe animal models followed by assays for related cytokinesand investigations of the IgM response involved

The results demonstrate that in the infant mouse modelIFKOS exhibited an inhibitory effect on pulmonary indexindicating that IFKOS protected the lung from MP-inducedinjury In the lung tissues of both infant mouse and ratmodels a large amount of pulmonary interstitial infiltrationof lymphocytes and macrophagocytes was observed as wellas significant inflammation cells around the bronchi whichindicated the characteristic pathological feature of MP pneu-monia [35] IFKOS effectively reduced pulmonary interstitialinfiltration of lymphocytes and macrophagocyte and thedegree of lesions in the lungs was significantly reduced inmouse and rat models This study therefore provides directevidence that IFKOS may represent a potential therapeuticagent for MP pneumonia

Anti-Mycoplasma pneumoniae-specific immunoglobulinM (MP-IgM) is an indicator of early acute infection thatcan persist for several months in patients MP-IgM typicallyappears within 1 week of the initial infection and approx-imately 2 weeks before MP-IgG so that elevation of MP-IgM alone can often be interpreted as evidence of acute

infection [36 37]We have detected the expression of specificMP-IgG in MP-mice model but the level of MP-IgG in theinfection group has no obvious change compared to thenormal control group IFKOS treatment (40 20 and 10mlkg)showed no significant effect on the level of MP-IgG Thepresence of IgM is considered most significant in pediatricpopulations and our mice model is in the early phase ofMP infection so the results of MP-IgG were not shown inthe manuscript Studies have demonstrated that the positiverate of IgM and IgA in Mycoplasma pneumoniae increasedto 975 and 563 respectively at one week after infectionCompared with MP-IgA MP-IgM has higher sensitivityin the diagnosis of mycoplasma-associated pneumonia innewborns school-aged children and adolescents [38 39]Mycoplasma pneumoniae affects the immune responsivenessof the host is their propensity for mitogenic stimulation ofB and T lymphocytes this property is associated with theability ofM pneumoniae to stimulate production of cytokinesand antibodies in the acute inflammatory response Theinhibitory effect of IFKOS on induction of specific MP-IgMmight be related to affect the function of B lymphocytes

Quantitative real-time PCR was used to investigate theability of MP to infect animals and the therapeutic effectof IFKOS The results showed that the relative expressionof MP-DNA in the lung tissues of the infection group wasobviously higher than in the lung tissues of the other groupsAZM treatment significantly reduced the relative expressionof MP-DNA compared with the infection group whereasIFKOS showed no obvious inhibitory effect on it The levelof MP-IgM in the infection group increased significantlycompared to the normal control group IFKOS treatmentobviously decreased the level ofMP-IgM in a dose-dependentmanner which indicated that elevation of MP-specific IgMwas significantly depressed by IFKOS treatment in a dose-dependent manner The results suggest that IFKOS stronglyinhibits acute infection induced by MP in infant mice themechanism may be related to the suppression of the IgMresponse induced by MP rather than directly inhibiting MPreplication

Some cases of mycoplasma pneumoniae pneumoniaresult in fulminant respiratory failure and are fatal in chil-dren [40] In a previous study the actual mortality rate infulminant MPP cases was reported to be 3ndash5 [41] In ourstudy the mortality rate survival rate death protection ratesurvival time and life-prolonging rate in BALBcmice modelwere calculated to estimate the protective effect of IFKOSon severe MP pneumonia The results demonstrated thatIFKOS treatment significantly decreased mortality rate anddramatically increased survival time in a dose-dependentmanner which indicated that IFKOS could protect infantmice from a lethal MP challenge

Excessive immune responses were involved in the patho-genesis of MP pneumonia especially in RMPP where thepathogenesis of pulmonary injury may be due to the hostrsquosimmune response rather than direct microbial damage [942] There is evidence to indicate that cytokines are closelyassociated with the development ofmycoplasma pneumoniaeinfections or the severity of disease both in patients andanimal models [43]


(a) (b) (c)

(d) (e) (f)

Figure 6 Pathological changes in lung tissues of Wistar rats After treatment with IFKOS for 4 days the right lobes of the lungs in rats werefixed in 10 paraformaldehyde embedded in paraffin and cut into slices for HampE staining Morphometric analysis was performed using anoptical microscope (a) Normal control group (b) infected control group (c) azithromycin treatment group (d) IFKOS (20mlkg) treatmentgroup (e) IFKOS (10mlkg) treatment group and (f) IFKOS (5mlkg) treatment group

However few studies of cytokines in Wistar rats havebeen reported So in order to investigate the role of cytokinesin the immunopathologic responses in MP pneumonia wedetected the changes of cytokines levels in lung tissues of anMP pneumonia rat model

IL-2 is a proinflammatory cytokine secreted from Th1cells and can promote a cell-mediated immune responsesto kill intracellular microbial pathogens [44] TNF-120572 is apolypeptide regulatory factor produced by Th1 cells withproinflammatory effects on eosinophils neutrophils T cellsand epithelial cells and it can cause local inflammationand even multiple organ injury [45] IL-6 is another impor-tant proinflammatory cytokine being a vital Th2 cytokineinvolved in the pathological process of pulmonary inflamma-tion It has been reported that IL-6 may be involved in theinfection process of MP and play an important role in thepathogenesis of MP pneumonia [46]

Our study showed that IL-2 and TNF-120572 levels signifi-cantly increased while the IL-6 level obviously decreased inthe lung tissues of rats infected with MP Of greater interestin the present study is that the elevated IL-2 and TNF-120572levels were significantly reduced by IFKOS treatment Inaddition the decreased IL-6 level was significantly enhancedby IFKOS at the doses of 10 and 5mlkgThe results indicatedthat proinflammatory cytokines IL-2 TNF-120572 and IL-6 weresimultaneously involved inMP pneumonia in the progress ofwhichTh1 cytokines (IL-2 and TNF-120572) were upregulated andTh2 cytokine (IL-6) was downregulated thereby changingthe balance of Th1Th2 toward Th1 cells A Th1Th2 balancefunction plays a significant role in anti-infectious immunityand the response of Th1Th2 cytokines are useful biomarkersin diagnosis and treatment of MP infection in children with

pulmonary involvement [47 48] Our studies demonstratedthat the imbalance of theTh1Th2 function afterMycoplasmapneumoniae infection is an important immunological mech-anism of MP pneumonia IFKOS treatment could reverse theimbalance of Th1Th2 cytokines induced by MP infection inthe infant rats It has been reported that IL-18 and IL-1szlig wereassociated with Mycoplasma pneumoniae infection We havedetected the levels of IL-18 and IL-1szlig in lung tissuesThe levelsof IL-18 and IL-1szlig were significantly increased in the infectiongroup compared with the normal control group but IFKOSshowed no obvious inhibitory effect on the elevated levels ofIL-18 and IL-1szlig So the results of IL-18 and IL-1szlig were notshowed in the manuscript

In conclusion our study shows that IFKOS has aninhibitory effect on Mycoplasma pneumoniae infection ininfant mouse and rat models of MP pneumonia and a pro-tective effect from lethal MP challenge in an infant murinemodelThese anti-MP effects might be related to suppressionof the IgM response and a reversal of the imbalance of Th1Th2 cytokines induced by MP infection These findings pro-vide direct evidence that IFKOS represents a potential propri-etary Chinese medicine to combat pediatric MP pneumonia

Data Availability

The research data used to support the findings of this studyare included within the article

Additional Points

Impact Statement In the present study the therapeutic effectand mechanisms of IFKOS on MP pneumonia were assessed


using infant mouse and rat models qRT-PCR and ELISAassay were used to evaluate the therapeutic effect of IFKOSon the expression levels of MP-DNA MP-IgM IL-2 IL-6 and TNF-120572 in response to MP pneumonia In additionpulmonary injury in mice and rats was detected by patho-logical examination under microscope and the mortality rateand survival time in BALBc mice model were observedOur study demonstrates that IFKOS has an inhibitory effecton MP infection in infant mouse and rat models of MPpneumonia and a protective effect from lethal MP challengein an infant murine model These anti-MP effects might berelated to suppression of the IgM response and a reversal ofthe imbalance ofTh1Th2 cytokines induced byMP infectionThese findings provide direct evidence that IFKOS representsa potential proprietary Chinese medicine to combat pediatricMP pneumonia

Conflicts of Interest

Theauthors do not have any conflicts of interest related to thiswork

Authorsrsquo Contributions

Shanshan Guo and Xiaolan Cui designed the study ShanshanGuo Lei Bao XinMao Yingjie Gao and Yingli Xu performedthe experiment Shanshan Guo Lei Bao and Tiange Quanalyzed and interpreted the data Shanshan Guo wrote themanuscript

Acknowledgments

This study was supported by manufacturer Kuihua Pharma-ceutical (China) and China Basic Research Projects of ChinaAcademy of Chinese Medical Sciences (No ZXKT15017academy batch nine)

References

[1] M Tashiro K Fushimi K Kawano et al ldquoAdjunctive corti-costeroid therapy for inpatients with Mycoplasma pneumoniaepneumoniardquo BMC Pulmonary Medicine vol 17 no 1 2017

[2] T Saraya ldquoMycoplasma pneumoniae infection Basicsrdquo Journalof General and Family Medicine vol 18 no 3 pp 118ndash125 2017

[3] T Ishida T Hashimoto M Arita Y Tojo H Tachibana andMJinnai ldquoA 3-year prospective study of a urinary antigen-detec-tion test for Streptococcus pneumoniae in community-acquiredpneumonia Utility and clinical impact on the reported etiol-ogyrdquo Journal of Infection and Chemotherapy vol 10 no 6 pp359ndash363 2004

[4] Z Shangguan Q Sun M Zhang et al ldquoMycoplasma pneumo-niae infection in hospitalized adult patients with community-acquired pneumonia in Chinardquo The Journal of Infection inDeveloping Countries vol 8 no 10 2014

[5] B Medjo M Atanaskovic-Markovic S Radic D Nikolic MLukac and S Djukic ldquoMycoplasma pneumoniae as a causativeagent of community-acquired pneumonia in children clinicalfeatures and laboratory diagnosisrdquo Italian Journal of Pediatricsvol 40 p 104 2014

[6] P M Meyer Sauteur W W Unger D Nadal C Berger CVink and A M van Rossum ldquoInfection with and Carriage of

Mycoplasma pneumoniae in Childrenrdquo Frontiers in Microbiol-ogy vol 7 2016

[7] M Narita ldquoPathogenesis of extrapulmonary manifestations ofMycoplasma pneumoniae infection with special reference topneumoniardquo Journal of Infection and Chemotherapy vol 16 no3 pp 162ndash169 2010

[8] Y Zhang S Mei Y Zhou M Huang G Dong and Z ChenldquoCytokines as the good predictors of refractory Mycoplasmapneumoniae pneumonia in school-aged childrenrdquo ScientificReports vol 6 2016

[9] MWang YWang Y Yan et al ldquoClinical and laboratory profilesof refractoryMycoplasma pneumoniae pneumonia in childrenrdquoInternational Journal of Infectious Diseases vol 29 pp 18ndash232014

[10] N Inamura N Miyashita S Hasegawa et al ldquoManagementof refractory Mycoplasma pneumoniae pneumonia Utilityof measuring serum lactate dehydrogenase levelrdquo Journal ofInfection and Chemotherapy vol 20 no 4 pp 270ndash273 2014

[11] K B Waites and D F Talkington ldquoMycoplasma pneumoniaeand its role as a human pathogenrdquo Clinical MicrobiologyReviews vol 17 no 4 pp 697ndash728 2004

[12] S Ding X Wang W Chen et al ldquoDecreased Interleukin-10Responses in Children with Severe Mycoplasma pneumoniaePneumoniardquo PLoS ONE vol 11 no 1 p e0146397 2016

[13] M Hayakawa H Taguchi S Kamiya et al ldquoAnimal Modelof Mycoplasma pneumoniae Infection Using Germfree MicerdquoClinical and Vaccine Immunology vol 9 no 3 pp 669ndash6762002

[14] T Saraya K Nakata K Nakagaki et al ldquoIdentification ofa mechanism for lung inflammation caused by Mycoplasmapneumoniae using a novel mouse modelrdquo Results in Immunol-ogy vol 1 no 1 pp 76ndash87 2011

[15] B Cao J Qu Y Yin and J V Eldere ldquoOverview of antimicrobialoptions for Mycoplasma pneumoniae pneumonia focus onmacrolide resistancerdquo The Clinical Respiratory Journal vol 11no 4 pp 419ndash429 2017

[16] H Lee K W Yun H J Lee and E H Choi ldquo Antimicrobialtherapy of macrolide-resistant rdquo Expert Review of Anti-infectiveTherapy vol 16 no 1 pp 23ndash34 2017

[17] Y-D Yin R Wang C Zhuo et al ldquoMacrolide-resistant Myco-plasma pneumoniae prevalence and clinical aspects in adultpatients with community-acquired pneumonia in China Aprospective multicenter surveillance studyrdquo Journal of ThoracicDisease vol 9 no 10 pp 3774ndash3781 2017

[18] K Cheong S S Chiu B W Chan K K To E L Chan andP Ho ldquoSevere macrolide-resistant Mycoplasma pneumoniaepneumonia associatedwithmacrolide failurerdquo Journal of Micro-biology Immunology and Infection vol 49 no 1 pp 127ndash1302016

[19] Q Qin B Xu X Liu and K Shen ldquoStatus of Mycoplasmapneumoniae Pneumonia in Chinese Children A SystematicReviewrdquo Advances in Microbiology vol 04 no 11 pp 704ndash7112014

[20] T Yamazaki and T Kenri ldquoEpidemiology of Mycoplasmapneumoniae infections in Japan and therapeutic strategies formacrolide-resistantM pneumoniaerdquo Frontiers in Microbiologyvol 7 no MAY 2016

[21] Y Yong-mei NMou and Z Ping ldquoimultaneous determinationof nine constituents in Xiaorsquoer Feire Kechuan Liquid by HPLC-DADrdquoChinese Traditional PatentMedicine vol 35 pp 709ndash7132013


[22] L Xiang L Guiyang M Jianli Z Liang andH Xinxin ldquoDeter-mination of Chlorogenic Acid in Xiaorsquoer Feire Kechuan OralSolutions by HPLCrdquo China Pharmacist vol 16 pp 1592-15932013

[23] R Meng ldquoObservation on the Effect of Xiaorsquoer Feire KechuanOral Solution with Azithromyci on Mycoplasma PneumoniaePneumoniardquo World Chinese Medicine vol 11 pp 1517ndash15192016

[24] Y Qiang and Y Haoyu ldquoXiaoer Feirekechuan Oral SolutionCombined with Azithromycin for Treating Pediatric Mycoplas-mal Pneumonia in 43 Casesrdquo China Pharmaceuticals vol 25pp 121-122 2016

[25] H Jin-fen L Xiao-yan and W Hui-juan ldquoEffect of azithro-mycin combined with infantile feirekechuan oral solution formycoplasma pneumonia in childrenrdquo Journal of XinxiangMedi-cal University vol 33 pp 483ndash488 2016

[26] I A Yoon K B Hong H J Lee et al ldquoRadiologic findings asa determinant and no effect of macrolide resistance on clini-cal course of Mycoplasma pneumoniae pneumoniardquo BMC In-fectious Diseases vol 17 no 1 article no 402 2017

[27] B Sztrymf F Jacobs J Fichet et al ldquoPneumopathie a myco-plasme une cause rare de syndrome de detresse respiratoireaigue (SDRA) et de resistance potentielle aux antibiotiquesrdquoRevue des Maladies Respiratoires vol 30 no 1 pp 77ndash80 2013

[28] Y Wang D Xu S Li and Z Chen ldquoMycoplasma pneumoniae-associated necrotizing pneumonitis in childrenrdquo PediatricsInternational vol 54 no 2 pp 293ndash297 2012

[29] H-J Yang D J Song and J Y Shim ldquoMechanism of resistanceacquisition and treatment of macrolide-resistant Mycoplasmapneumoniae pneumonia in childrenrdquo Korean Journal of Pedi-atrics vol 60 no 6 pp 167ndash174 2017

[30] N Principi and S Esposito ldquoMacrolide-resistant Mycoplasmapneumoniae Its role in respiratory infectionrdquo Journal of Antimi-crobial Chemotherapy vol 68 no 3 pp 506ndash511 2013

[31] Mei Jiang-hua ldquoClinical Observation on 45 Cases of Chil-dren with Mycoplasma Pneumoniae Mycoplasma PneumoniaTreated by Sequential Azithromycin Combined with infantilefeirekechuan oral solutionrdquo Chinese Remedies amp Clinics pp1197-1198 2016

[32] Q Song B Xu and K Shen ldquoBacterial co-infection in hos-pitalized children with Mycoplasma pneumoniae pneumoniardquoIndian Pediatrics vol 53 no 10 pp 879ndash882 2016

[33] A-J Yang K Xi Y-Q Yao Y-X Ding L-J Yang and HCui ldquoEffect of Qingfei Mixture () on pediatric mycoplasmapneumoniae pneumonia with phlegm heat obstructing Fei(Lung) syndromerdquo Chinese Journal of Integrative Medicine vol23 no 9 pp 681ndash688 2017

[34] Y Jiang J Yu A Guo et al ldquoAmeliorative effects of QingfeiTongluo formula on experimental mycoplasmal pneumonia inmicerdquo Journal of Natural Medicines vol 70 no 2 pp 145ndash1512016

[35] F Gally M N Minor S K Smith S R Case and H W ChuldquoHeat shock factor 1 protects against lung Mycoplasma pneu-moniae infection in micerdquo Journal of Innate Immunity vol 4no 1 pp 59ndash68 2011

[36] B Medjo M Atanaskovic-Markovic S Radic D Nikolic MLukac and S Djukic ldquoMycoplasma pneumoniae as a causativeagent of community-acquired pneumonia in children clinicalfeatures and laboratory diagnosisrdquo Italian Journal of Pediatricsvol 40 no 1 2014

[37] D F Talkington S Shott M T Fallon S B Schwartz andW LThacker ldquoAnalysis of eight commercial enzyme immunoassay

tests for detection of antibodies to Mycoplasma pneumoniae inhuman serumrdquoClinical andDiagnostic Laboratory Immunologyvol 11 no 5 pp 862ndash867 2004

[38] W Lee E Huang C Tsai et al ldquoRole of Serum Mycoplasmapneumoniae IgA IgM and IgG in theDiagnosis ofMycoplasmapneumoniae-Related Pneumonia in School-Age Children andAdolescentsrdquo Clinical and Vaccine Immunology vol 24 no 12017

[39] Y Dong W Lv and Z Lin ldquoValue of serum Mycoplasmapneumoniae immunoglobulin in the diagnosis of mycoplasma-relatedpneumonia in newbornsrdquo Experimental andTherapeuticMedicine vol 14 no 2 pp 1445ndash1449 2017

[40] Y Zhang Y Zhou S Li et al ldquoThe Clinical Characteristics andPredictors of Refractory Mycoplasma pneumoniae Pneumoniain Childrenrdquo PLoS ONE vol 11 no 5 p e0156465 2016

[41] K Izumikawa ldquoClinical Features of Severe or FatalMycoplasmapneumoniae Pneumoniardquo Frontiers inMicrobiology vol 7 2016

[42] S Kurata T Osaki H Yonezawa K Arae H Taguchi and SKamiya ldquoRole IL-17A and IL-10 in the antigen induced inflam-mation model by Mycoplasma pneumoniaerdquo BMC Microbiol-ogy vol 14 no 1 article no 156 2014

[43] X-F Xu X-J Li J-L Liu L Wu and Z-M Chen ldquoSerumcytokine profile contributes to discriminating M pneumoniaepneumonia in childrenrdquo Cytokine vol 86 pp 73ndash78 2016

[44] MAkdis A Aab C Altunbulakli et al ldquoInterleukins (from IL-1to IL-38) interferons transforming growth factor 120573 and TNF-120572 Receptors functions and roles in diseasesrdquo The Journal ofAllergy and Clinical Immunology vol 138 no 4 pp 984ndash10102016

[45] G M Walsh ldquoAntagonism of cytokine-induced eosinophilaccumulation in asthmardquo Frontiers in Pharmacology vol 32012

[46] J He M Liu Z Ye et al ldquoInsights into the pathogenesis ofMycoplasma pneumoniaerdquoMolecularMedicine Reports vol 14no 5 pp 4030ndash4036 2016

[47] W Li Y-J Liu X-L Zhao et al ldquoTh1Th2 cytokine profile andits diagnostic value in Mycoplasma pneumoniae pneumoniardquoIranian Journal of Pediatrics vol 26 no 1 2016

[48] J Zhao XWang andYWang ldquoRelationships betweenTh1Th2cytokine profiles and chest radiographic manifestations inchildhoodMycoplasma pneumoniae pneumoniardquoTherapeuticsand Clinical Risk Management vol Volume 12 pp 1683ndash16922016

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


found that IL-8 IL-10 and IL-18 appeared to modulatepulmonary lesions inMP pneumonia in children of a medianage of 5 years [12] In addition the production of IL-2 IL-4IL-10 and IFN-120574 by splenic lymphocytes has been detectedin significant high levels in a gnotobiotic mice model ofMycoplasma pneumoniae pneumonia and IL-1szlig and IL-6were upregulated in a novel mouse model immunized withMP extracts plus alum [13 14]

Macrolides have been recommended as the first-linetherapy in adults whereas azithromycin (AZM) as thefirst-line drug in for pediatric cases [15 16] Howevermacrolide-resistant MP pneumoniae (MRMP) pneumoniahave emerged and became widespread in East Asia since2000 In the last decade macrolide resistance rates in MRMPpneumoniae among children have substantially increasedworldwide [17 18] especially inChina and Japan where it hasreached up to 90 [19 20] Therefore it is extremely urgentto develop new drugs as safe and effective alternative agentsfor the treatment of MP pneumonia

Infantile Feire Kechuan Oral Solution (IFKOS) is atraditional Chinese medicine that is mainly used to treatrespiratory tract infections such as influenza and pneumoniain children IFKOS is composed of the following Chinesemedicines Lonicerae japonicae Flos Ephedrae Herba Gly-cyrrhizae Radix et Rhizoma Forsythiae Fructus Scutellar-iae Radix and Armeniacae Semen amarum Studies havereported that active constituents of IFKOS such as chloro-genic acid and forsythiaside may have pharmacological func-tions of wind dispelling heat-clearing detoxifying and painrelieving [21 22] In China IFKOS has been listed as a pro-prietary Chinese medicine to combat pediatric respiratoryinfection Many clinical studies have reported the curativeeffects of IFKOS against MP pneumonia and the cure ratewas up to 9070 when infantile MP pneumonia was treatedwith IFKOS combined with azithromycin [23ndash25] Howeverfew studies have investigated the curative and underlyingmechanisms of the actions involved in experimental models

In the present study the therapeutic effect and mech-anisms of IFKOS on MP pneumonia were assessed usinginfancy BALBc mouse and Wistar rat models Quantitativereal-time polymerase chain reaction and an enzyme-linkedimmunosorbent assay (ELISA) were used to evaluate thetherapeutic effect of IFKOS on the expression levels of MP-DNA MP-IgM IL-2 IL-6 and TNF-120572 in response to MPpneumonia In addition pulmonary injury in mice and ratswas detected by pathological examination under microscopeand the mortality rate and survival time in a BALBc micemodel were observed

2 Materials and Methods

21 Test Drugs All investigations were performed with asingle batch (No 201610001) of IFKOS kindly provided by themanufacturer Kuihua Pharmaceutical (Heilongjiang China)The doses of IFKOS were 40 20 and 10mlkgday for infantmice and 20 10 and 5mlkgday for infant rats All the dosesfor mice and rats were equivalent to two one and half timesthe clinical dose of 6-year-old children (2mlkg) accordingto the ratio of body surface area of human and mice or rats

respectively Azithromycin (batch No 063160821) was pro-duced by Ouyi Pharma China Shijiazhuang PharmaceuticalGroup served as a reference drug The therapeutic dose ofazithromycin is 5 mgkg daily (15-25kg children) and thedoses for mice and rats were 50 and 25mgkgday accordingto the ratio of body surface area of human and mice or ratsrespectively

22 Animals Male and female BALBc mice (13-15g) andWistar rats (90-110g) were purchased from Vital River Lab-oratory Animal Technology Co Ltd in Beijing China(Certificate No SCXK 2016-0011) and maintained in anenvironmentally controlled breeding room (temperature 20plusmn 2∘C humidity 50 plusmn 10) The study was carried out inaccordance with the rules of European Community Guide-lines for care and use of animals The study was approvedby Ethics Committee at Institute of Chinese Materia MedicaChina Academy of Chinese Medical Sciences

23 MP Culture MP FH strain (ATCC15531 AmericanType Culture Collection Rockville MD USA) was culturedin modified mycoplasma medium (Oxoid UK) containingPPLO broth horse serum and 25 yeast extract to whichwas added penicillin G (200 Uml) thallium acetate (0025) glucose (1 ) and phenol red (0002 ) pH 78 MP wascultured at 37∘Cunder 5 CO

2for 7 daysThe concentration

of MP was determined as 108 color change units (CCU)mlaccording to the color-change-unit method [26]

24 Mouse Model of MP Pneumonia BALBc mice wererandomly divided into six groups with 10 mice per group(half male and half female) The groups were assignedas normal control group infection control group andfour treatment groups (three IFKOS treatment groups andone positive drug-azithromycin treatment group) All miceexcept those in the normal control group were repeatedlyinfected intranasally with 50120583L of the MP solution at aconcentration of 108CCUml for three times once a day Inthe treatment groups the inoculated mice were successivelygiven IFKOS orally at a dose of 40mlkg 20mlkg or 10mlkgor treated with azithromycin at a dose of 50mgkg daily for3 days beginning from the day of infection Each mouse ofthe control group was given an equal volume of distilledwater On day 4 blood of all the mice was collected andthe serum was separated to determine the level of MP-IgM Then the mice were sacrificed and the body weight(BW) and lung weight (LW) were measured to calculatethe pulmonary index (PI) and inhibitory rate of pulmonaryindices (IRPI)PI=[LW(g)BW(g)]times100IRPI=[(LW of infec-tion control group-LW of treatment group)(LW of infectioncontrol group-LW of normal control group)]times100 The lungtissues were extracted and subjected to qRT-PCR detectionand histological examination

25 Rat Model of MP Pneumonia Wistar rats were ran-domly divided into six groups with 10 rats per group (halfmale and half female) as described above All rats exceptthose in the normal control group were repeatedly infectedintranasally with 150120583L of the MP solution at a concentration

















3 Results



5

45

4

35

3

25

2

15

05

0

1


group

rela

tive e

xpre

ssio

n of

MP-

DN

D(lo

g va

lue o

f cop

oes)

(a)


group

180

160

140

120

100

80

60

40

20

0leve

l of M

P-Ig

M (n

gm

l)

(b)

















(a) (b) (c)

(d) (e) (f)


(a) (b) (c)

(d) (e) (f)



100

80

60

40

20

00 12108642



Perc

ent s

urvi

val

(a)

0

12

10

8

6

4

2 surv

ival

tim

e (da

y)


(b)


250

200

150

100

50

0

leve

l of T

NF-

(n

gm

l)


group

(a)

300

250

200

150

100

50

0

leve

l of I

L-2

(ng

ml)


group

(b)

600

500

400

300

200

100

0

leve

l of I

L-6

(ng

ml)


group

(c)







4 Discussion











(a) (b) (c)

(d) (e) (f)







Data Availability


Additional Points








Acknowledgments


References
























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



































3 Results



5

45

4

35

3

25

2

15

05

0

1


group

rela

tive e

xpre

ssio

n of

MP-

DN

D(lo

g va

lue o

f cop

oes)

(a)


group

180

160

140

120

100

80

60

40

20

0leve

l of M

P-Ig

M (n

gm

l)

(b)

















(a) (b) (c)

(d) (e) (f)


(a) (b) (c)

(d) (e) (f)



100

80

60

40

20

00 12108642



Perc

ent s

urvi

val

(a)

0

12

10

8

6

4

2 surv

ival

tim

e (da

y)


(b)


250

200

150

100

50

0

leve

l of T

NF-

(n

gm

l)


group

(a)

300

250

200

150

100

50

0

leve

l of I

L-2

(ng

ml)


group

(b)

600

500

400

300

200

100

0

leve

l of I

L-6

(ng

ml)


group

(c)







4 Discussion











(a) (b) (c)

(d) (e) (f)







Data Availability


Additional Points








Acknowledgments


References
























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















5

45

4

35

3

25

2

15

05

0

1


group

rela

tive e

xpre

ssio

n of

MP-

DN

D(lo

g va

lue o

f cop

oes)

(a)


group

180

160

140

120

100

80

60

40

20

0leve

l of M

P-Ig

M (n

gm

l)

(b)

















(a) (b) (c)

(d) (e) (f)


(a) (b) (c)

(d) (e) (f)



100

80

60

40

20

00 12108642



Perc

ent s

urvi

val

(a)

0

12

10

8

6

4

2 surv

ival

tim

e (da

y)


(b)


250

200

150

100

50

0

leve

l of T

NF-

(n

gm

l)


group

(a)

300

250

200

150

100

50

0

leve

l of I

L-2

(ng

ml)


group

(b)

600

500

400

300

200

100

0

leve

l of I

L-6

(ng

ml)


group

(c)







4 Discussion











(a) (b) (c)

(d) (e) (f)







Data Availability


Additional Points








Acknowledgments


References
























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a) (b) (c)

(d) (e) (f)


(a) (b) (c)

(d) (e) (f)



100

80

60

40

20

00 12108642



Perc

ent s

urvi

val

(a)

0

12

10

8

6

4

2 surv

ival

tim

e (da

y)


(b)


250

200

150

100

50

0

leve

l of T

NF-

(n

gm

l)


group

(a)

300

250

200

150

100

50

0

leve

l of I

L-2

(ng

ml)


group

(b)

600

500

400

300

200

100

0

leve

l of I

L-6

(ng

ml)


group

(c)







4 Discussion











(a) (b) (c)

(d) (e) (f)







Data Availability


Additional Points








Acknowledgments


References
























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















100

80

60

40

20

00 12108642



Perc

ent s

urvi

val

(a)

0

12

10

8

6

4

2 surv

ival

tim

e (da

y)


(b)


250

200

150

100

50

0

leve

l of T

NF-

(n

gm

l)


group

(a)

300

250

200

150

100

50

0

leve

l of I

L-2

(ng

ml)


group

(b)

600

500

400

300

200

100

0

leve

l of I

L-6

(ng

ml)


group

(c)







4 Discussion











(a) (b) (c)

(d) (e) (f)







Data Availability


Additional Points








Acknowledgments


References
























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















4 Discussion











(a) (b) (c)

(d) (e) (f)







Data Availability


Additional Points








Acknowledgments


References
























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a) (b) (c)

(d) (e) (f)







Data Availability


Additional Points








Acknowledgments


References
























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

























Acknowledgments


References
























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















ameliorative effects of infantile feire kechuan oral

Documents