original article hepatoprotective activity of pien tze huang gan...

Int J Clin Exp Med 2019;12(9):11324-11332www.ijcem.com /ISSN:1940-5901/IJCEM0090165

Original Article Hepatoprotective activity of Pien Tze Huang Gan Bao in CCl4-induced chronic liver injury models

Jinyan Zhao1,2, Daxin Chen1,2, Shan Lin1,2, Yuchen Zhang1,2, Yun Wan1,2, Zhenfeng Hong1

1Academy of Integrative Medicine, 2Fujian Key Laboratory of Integrative Medicine on Geriatric, Fujian University of Traditional Chinese Medicine, Qiuyang Road, Shangjie Minhou, Fuzhou 350122, Fujian, China

Received December 20, 2018; Accepted April 9, 2019; Epub September 15, 2019; Published September 30, 2019

Abstract: The aim of the current study was to evaluate the effects and underlying molecular mechanisms of Pien Tze Huang Gan Bao (GB) on chronic liver injuries induced by carbon tetrachloride (CCl4) in rat models. Chronic liver inju-ries in Sprague-Dawley male rats were induced by intraperitoneal injections with CCl4 (1.0 mL/kg body weight, twice weekly), dissolved in olive oil (1:1, v/v). After four weeks, experimental and control rats were orally administered physiological saline (PS), silymarin (50 mg/kg), GB-Low (150 mg/kg), GB-moderate (300 mg/kg), or GB-high (600 mg/kg) for 4 weeks. Twenty-four hours after the last treatment, the animals were sacrificed. Biochemical assays were used to analyze indicators of hepatotoxicity. H&E staining was employed to observe histopathological changes in the liver and Masson’s staining was used to assess liver fibrosis. Gene and protein expression levels of TGF-β1, α-SMA, IL-1β, and TNF-α in the liver were determined by qPCR and ELISA or Western blotting, respectively. Results showed that both silymarin and GB normalized elevated levels of ALT, AST, ALP, and LDH and attenuated hepatic steatosis and fibrosis induced by CCl4. GB treatment also decreased secretion of LN and HA and downregulated expression of TGF-β1, α-SMA, IL-1β, and TNF-α. GB demonstrated protective effects against chronic liver injuries induced by CCl4, likely through the alleviation of inflammation-induced hepatic fibrosis.

Keywords: Pien Tze Huang Gan Bao, carbon tetrachloride, liver injury, liver fibrosis, inflammation

Introduction

The liver is an important metabolic organ in the body, though it is susceptible to injury by vari-ous factors, including many chemical agents. Chronic liver disease represents a major public health concern, worldwide. Hepatocellular apoptosis, hepatic inflammation, oxidative stress, and fibrosis are prominent features of chronic liver disease [1, 2]. Of these, progressive hepat-ic fibrosis is the common pathway of most chronic liver injuries, leading to liver cirrhosis and hepatocellular carcinoma [3]. Carbon tetra-chloride (CCl4)-induced liver injury is a classic model of chemical liver injury in rodents [4-6], popular for its symptoms resembling those of chronic liver injury in humans [7]. Despite advances in the understanding of the molecu-lar pathology of liver damage, there are only a limited number of hepatoprotective interven-tions. In view of this, many have posited the potential of natural compounds, which have

demonstrated worldwide importance and effec-tiveness [8].

Natural remedies from medicinal plants are considered safe and effective alternatives or complementary treatments against hepatotox-icity. Pien Tze Huang Gan Bao (GB), a Traditional Chinese Medicine containing Calculus bovis, Panax notoginseng, Artemisia capillaris, snake gall, and Radix Paeoniae alba, has been used for millennia in China and Southeast Asia. GB has been demonstrated to be an effective pro-tectant against liver injuries caused by exces-sive alcohol consumption. However, there re- mains a lack of information concerning the molecular mechanisms by which GB confers protective actions. The present study estab-lished a chronic liver injury model via CCl4 in rats, aiming to investigate the underlying mech-anisms of the protective roles of GB in chronic liver injuries. This study was conducted with an emphasis on attenuation of inflammation and fibrosis.

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Pien Tze Huang Gan Bao alleviates hepatic fibrosis

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Methods and materials

GB was obtained and authenticated by the sole manufacturer, Zhangzhou Pien Tze Huang Pharmaceutical Co. Ltd. (Zhangzhou, China; Chinese FDA approval no. HPK-08411). Si- lymarin (cat no: 02000585) was obtained from Sigma-Aldrich Company (Sigma-Aldrich; Merck KGaA). TRIzol Reagent was purchased from Life Technologies (Thermo Fisher Scientific, Inc., Waltham, MA, USA). PrimeScript™ RT reagent kit with gDNA Eraser and SYBY Premix Ex Taq II (Tli RNaseH plus) was purchased from Takara Bio Inc., (Tokyo, Japan). BCA Protein Assay Kit was purchased from Tiangen Biotech Co., Ltd., (Beijing, China). Laminin (LN), Hyaluronic acid (HA), Interleukin (IL)-1β, and Tumor necrosis factor (TNF)-α enzyme linked immunosorbent (ELISA) assay kits were purchased from Shanghai Xitang Biotech Co., Ltd. (Shanghai, China). GAPDH (5174S; 1:1,000 dilution) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit monoclonal anti-bodies for alpha smooth muscle actin (α-SMA, cat no. ab32575) and rabbit polyclonal to trans-forming growth factor beta 1 (TGF-β1, cat no. ab92486) were obtained from Abcam (Abcam, Cambridge, MA, USA; 1:1,000 dilution). Ho- rseradish peroxidase-conjugated secondary antibodies (cat. no. ab205718) were obtained from Abcam (1:2,000 dilution). CCl4 was pur-chased from Shanghai Lingfeng Chemical Co., Ltd. (Shanghai, China).

Animals

Sixty male Sprague-Dawley rats (6 weeks; 180-200 g; Slike Co., Ltd., Shanghai, China) were grouped with five in one cage (ventilated) in an environmentally controlled facility. Temperature was maintained at 22 ± 1°C and 40-60% humidity. The housing area was kept under a 12-hour light/dark cycle, with a light intensity of around 150-300 lux. Food and water were provided ad libitum for one week prior to the beginning of experimentation. All animal stud-ies were approved by the Fujian Institute of Traditional Chinese Medicine Animal Ethics Committee (Fuzhou, China). Experimental pro-cedures were carried out in accordance with Guidelines for Animal Experimentation of Fu- jian University of Traditional Chinese Medicine (Fuzhou, China).

Chronic liver injuries were induced by intraperitoneal injections of CCl4 (1.0 mL/kg body

weight, twice weekly), dissolved in olive oil (1:1, v/v), for 8 weeks. At the beginning of treatment, the animals were randomly assigned into six groups (n = 10, each), according to the following conditions: Group 1: Control group; Group 2: CCl4 model group; Group 3: Silymarin treated CCl4 group (treated with silymarin, 50 mg/kg), a positive control; Group 4: Low-dose GB treat-ment CCl4 group (treated with GB, 150 mg/kg); Group 5: Medium-dose GB treatment CCl4 group (treated with GB, 300 mg/kg); and Group 6: High-dose GB treatment CCl4 group (treated with GB, 600 mg/kg). On the fifth week, Groups 3 to 6 were administered oral doses of silyma-rin or GB for an additional four weeks. Instead, control and CCl4 model groups were given oral doses of PS for 4 weeks. Twenty-four hours after the final treatment, the rats were anesthe-tized by 40 mg/kg intraperitoneal injections of pentobarbital (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Once anesthesia con-firmed, the animals were terminated by cervical dislocation. They were immediately subjected to laparotomy procedures, in which the abdomi-nal cavity was opened to expose the abdominal aorta. Blood was collected from the aorta abdominalis into non-heparinized tubes. These tubes were centrifuged at 3,000 rpm at 4°C for 10 minutes, obtaining serum for biochemical testing and ELISA assays. Liver tissues were also quickly excised into multiple portions and washed. This was followed by fixation in formal-dehyde saline (4%) solution for histological analysis. The remaining portion was snap-fro-zen in liquid nitrogen and stored at -80°C for molecular analysis.

Serum biochemical assays

Various markers of hepatic function, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH), were evaluated using an automatic biochemical analyzer (Bayer ADVIA 2400; Bayer, Siemens, Germany), according to manufacturer instructions.

Histopathological analysis

Liver tissues allocated for histopathological analysis were fixed in 10% buffered formalin for ≥ 48 hours. This was followed by process- ing and paraffin-embedding. Paraffin sections were prepared by an automatic tissue proces-


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sor (RM2; Leica Microsystems GmbH, Wetzlar, Germany) and sectioned into 5 µm-thick serial slices using a rotary microtome. Sections were subsequently stained by hematoxylin-eosin dye and histopathologically analyzed. Photomicro- graphs of the stained tissues were captured using a DMRB/E light microscope (Leica Mi- crosystems GmbH).

Masson’s trichrome staining

The hepatic tissue sections were deparaffiniz- ed, dehydrated, washed, and stained in Wei- gert’s iron hematoxylin for 5 minutes. Next, they were washed and stained in Biebrich scar-let acid fuchsin solution for 5 minutes. The sec-tions were then washed and differentiated in 1% phosphomolybdic-phosphotungstic acid solution for 5 minutes. This was followed by ani-line blue counterstain for 5 minutes. Finally, the sections were differentiated in 1% acetic acid solution for 1 minute, followed by washing, dehydration, and treatment with resin mount-ing medium.

ELISA

Commercial enzyme-linked immunosorbent assays (ELISA) were used for evaluation of various markers, including LN, HA, TNF-α, and IL-1β. The kits operated on a solid-phase sandwich principle, beginning with the addition of a monoclonal antibody specific for rat LN, HA, TNF-α, or IL-1β coated on 96-well plates. Standards and samples were incubated in antibody-con-taining solution, allowing for the binding of any present LN, HA, TNF-α, or IL-1β to the immobi-lized antibody. Plates were washed and a sec-ondary biotinylated polyclonal anti-rat LN, HA, TNF-α, or IL-1β antibody was added. Finally, avidin-horseradish peroxidase tertiary antibod-ies were added as the last sequence in the antibody-antigen-antibody sandwich formation. To visualize positive signals, a substrate solu-tion was added, generating a blue color propor-tional to the amount of rat LN, HA, TNF-α, or IL-1β present in the sample. A stop buffer was next added to terminate the reaction. Ab- sorbance values of each reaction were then measured at 450 nm on a plate reader. Optical density levels were converted to pg/mg of pro-tein for each amount of rat LN, HA, TNF-α, or IL-1β.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total liver RNA was isolated using TRIzol Re- agent, according to manufacturer instructions. Purified RNA was quantified by measuring the absorbance of the preparation at 260 nm. Moreover, cDNA synthesis was performed using the PrimeScript™ RT reagent kit, according to manufacturer instructions. One microgram of total RNA was incubated with 4.0 μl 5X Pri- meScript buffer, 1.0 μl PrimeScript RT enzyme mix I, 1.0 μl Oligo dT primer (50 μM), 1.0 μl ran-dom 6 mers (100 μM), and RNase-free water to a final volume of 20 μl. The reaction mixture was processed at 25°C for 10 minutes. It was heated to 37°C for 15 minutes, heated to 85°C for 5 seconds, and cooled to 4°C.

Next, cDNA was subjected to PCR amplification using SYBR Premix Ex Taq II in an ABI 7500 instrument. Expression of mRNA was deter-mined using the 2-∆∆Cq method [9], based on the expression of the internal control gene for β-actin. All qPCR reactions were conducted in triplicate. Primer sequences were as follows: IL-1β forward, 5’-TGT TCT TTG AGG CTG AC-3’ and reverse, 5’-CTT TGG GAT TTG TTT GG-3’; TNF-α forward, 5’-CAG CAG ATG GGC TGT ACC TT-3’ and reverse, 5’-AAG TAG ACC TGC CCG GAC TC-3’; TGF-β1 forward, 5’-TGA ACC AAG GAG ACG GAA TAC AGG-3’ and reverse, 5’-GCA GTA GTT GGT ATC CAG GGC TCT-3’, α-SMA for-ward, 5’-ATC ACC ATC GGG AAT GAA CGC TT-3’ and reverse, 5’-ATC CTG TCA GCA ATG CCT GGG TA-3’; and β-actin forward, 5’-CGG TCA GGT CAT CAC TAT CGG C-3’ and reverse, 5’-GTG TTG GCA TAG AGG TCT TTA CG G-3’.

Western blot analysis

Livers tissues were mechanically homogenized and total protein was extracted by radioimmu-noprecipitation using a commercial preparation (Thermo Fisher Scientific, Inc.), containing protease and phosphatase inhibitor cocktails. After homogenization, protein lysate was cen-trifuged at 12,000 × g for 15 minutes at 4°C. Protein concentrations were determined by the BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Fifty microgram samples were separated by 10% SDS-PAGE and transfer- red onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The mem-


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branes were incubated overnight at 4°C with blocking solution in TBS, followed by primary antibody incubation (rabbit monoclonal anti-bodies against TGF-β1, α-MSA, and GAPDH) for 2 hours at room temperature. Subsequently, the membranes were washed and incubated with an anti-rabbit horseradish peroxidase-con-jugated secondary antibody (1:2,000) for 1 hour at room temperature. To visualize pro-teins, the membranes were washed a final time in TBST solution. This was followed by the addition of SuperSignal™ West Pico Chemilu- minescent Substrate (Thermo Fisher Scienti- fic, Inc.) for enhanced chemiluminescence de- tection.

High performance liquid chromatography (HPLC)

The samples were analyzed on an Agilent 1200 HPLC system (Agilent Technologies, San- ta Clara, CA, USA) using a Welch Ultimate XB-C18 (250 mm × 4.60 mm; 5 μm). Absorban- ce was measured at 274 nm. The mobile phase consisted of methanol and 0.1% phosphoric acid (45:55) at a flow rate of 0.9 mL/min, with an injection volume of 5 μl. Column tempera-ture was maintained at 20°C. Baicalin (Sigma-Aldrich; Merck KGaA) served as a positive con-trol [10].

the control group. Elevation in secretion levels of these enzymes was significantly decreased (P < 0.01) by silymarin and 600 mg/kg of GB, compared to the CCl4 group, as shown in Figure 1. Similarly, serum LDH was increased in CCl4-treated animals, an effect reversed by sily-marin- or GB-treatment. Treatment with 150, 300, and 600 mg/kg PZH-GB, as well as sily-marin at a dose of 50 mg/kg, exhibited a reduc-tion of in serum LDH by 44.30%, 31.90%, 45.17%, and 59.53%, respectively.

GB ameliorates hepatic gross morphology and histopathological changes

Hepatic gross morphology was captured using a single lens reflex camera (Nikon, Japan). While control rats exhibited a normal morphol-ogy (capsule smooth, complete, red-brown red, and a soft texture), livers in the CCl4 group showed typical hepatic fibrosis morphology, with a yellowed rough surface, blunt edge, fiber exudation, and adhesion with surrounding tis-sues. In contrast, after treatment with silymarin and GB (especially 600 mg/kg), the livers showed obvious signs of recovery (Figure 2A). Furthermore, liver histopathology was analyzed with H&E staining, revealing a lobular architec-ture and hepatic cells with well-preserved cyto-plasm, prominent nucleus and nucleolus, visi-ble central veins, and thin sinusoids in the con-

Figure 1. Effects of GB on ALT, AST, ALP, and LDH after CCl4 injury. #P < 0.05, vs. control group; *P < 0.05 vs. CCl4-model group. n = 10 in each group; Values are mean ± S.D. GB, Pien Tze Huang Gan Bao; ALT, alanine amino-transferase; AST, artate aminotransferase; ALP, alkaline phosphatase; LDH, lactate dehydrogenase; CCl4, carbon tetrachloride; GB-L, GB-M, GB-H, PZH-GB low, medium, and high-dose treatment groups, respectively.

Statistical analysis

Data were analyzed using SP- SS version 11.5 (SPSS, Inc., Chicago, IL, USA) and are ex- pressed as mean ± standard deviation of three independent experiments. Statistical analy-sis was performed with one-way analysis of variance, fol-lowed by post-hoc Fisher’s least significant difference testing. P < 0.05 indicates statis-tically significant differences.

Results

GB prevents CCl4-induced in-creases in hepatic enzymes

Administration of CCl4 marked-ly increased (P < 0.01) the activity of hepatic enzymes AST, ALT, and ALP, compared to


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trol group (Figure 2B). In contrast, the injury group showed extensive damage, exhibiting hepar arranged messily, broad inflammatory cell infiltration, ballooning, degeneration, and loss of cellular boundaries. Liver sections of rats treated with GB (150, 300, and 600 mg/kg) or silymarin, on the other hand, suggested that treatment noticeably reduced or prevent-ed the development of histopathological dam-age, particularly in the 600 mg/kg GB group (Figure 2B).

GB prevented liver injuries via attenuation of fibrosis

Liver fibrosis was evaluated histologically via Masson’s staining. Results demonstrated that normal hepatic lobules in the CCl4 group were diminished and collagen deposits were evi-dent. Treatment with silymarin and GB mark-edly alleviated collagen deposition and lowered

levels of fibrosis, compared to the CCl4 group (Figure 3A). To confirm the anti-fibrosis effects of silymarin and GB, expression levels of com-mon liver fibrosis biomarkers HA and LN were determined in the serum. HA in the injury group was elevated by 39.76%, while LN was increased 39.02%, compared with the control group. Treatment with silymarin and different doses of GB reduced serum levels of HA and LN. Compared with the induced injury model, silymarin and GB (150, 300, and 600 mg/kg) decreased levels of LN or HA by 60.67, 36.89, 30.39, and 49.68% or 35.34, 24.10, 36.55, and 36.55%, respectively (Figure 3B). Eva- luation of signaling molecules TGF-β1 and α-SMA revealed that GB (especially 600 mg/kg group) inhibited TGF-β1 and α-SMA (Figure 4). Taken together, present data demonstrates that silymarin and GB ameliorated liver fibrosis, indicated histologically by serum biomarkers

Figure 2. GB administration ameliorates hepatic gross morphology and histopathological changes. A. Livers from the injury group show typical hepatic fibrosis patterns, with more yellow, rough surface, blunt edge, fiber exudation, and adhesion with surrounding tissues. GB or silymarin treated groups show improved hepatic gross morphology; B. Hematoxylin and eosin (HE) staining of liver sections show CCl4 induced broad inflammatory cell infiltration and ballooning degeneration, while GB or silymarin treatments reversed this effect. The photographs are representative images taken at a magnification of × 200.


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and expression of TGF-β1 and α-SMA.

GB downregulated expression of pro-inflammatory factors

Pro-inflammatory cytokines are known to play a crucial role in liver injuries and fibrosis [11-14]. The present study exam-ined the effects of GB on pro-inflammatory gene expression of chronic liver-injury rats, spe-cifically by measuring TNF-α and IL-1β, using RT-qPCR, in liver tissues (Figure 5A). Re- sults showed that significant increases in TNF-α and IL-1β mRNA occurred in the livers of CCl4-treated rats. As shown in Figure 5A, CCl4 intoxication increased the mRNA of TNF-α and IL-1β by 42.29% or 29.08%, respectively. However, treatment with GB and silymarin pre-vented these expression level

Figure 3. GB administration relieves hepatic fibrosis induced by CCl4. A. Masson’s staining analysis of hepatic fi-brosis; Photographs are representative images taken at a magnification of × 200; B. Effects of GB on FN and LA in serum. #P < 0.05, vs. control group; *P < 0.05 vs. CCl4-model group. n = 10 in each group; Values are mean ± S.D.

Figure 4. GB downregulates expression of TGF-β1 and α-SMA in the liver. (A) mRNA and (B) Protein expression levels of TGF-β1 and α-SMA, as deter-mined by reverse transcription-quantitative polymerase chain reaction and Western blotting. GAPDH served as the internal control. Data are presented as the mean ± standard deviation. (C) Quantification of TGF-β1 and α-SMA protein. #P < 0.05 vs. control group; *P < 0.05 vs. model group.


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increases in the pro-inflammatory cytokines. Inhibition of TNF-α and IL-1β gene expression by silymarin and GB was further confirmed by measurement of TNF-α and IL-1β protein using ELISA assays (Figure 5B). TNF-α and IL-1β pro-tein was elevated by CCl4 but inhibited by either GB or silymarin.

Discussion

Liver injury models induced by CCl4 are most commonly used for screening the hepatopro-tective activity of natural substances [15-17]. The current study induced chronic liver injuries in rats via intraperitoneal injections of CCl4 for 8 weeks. This was based on previous data showing that CCl4 at a dose of 1 mL/kg of body weight, twice per week for 8 weeks, was enough for development of hepatic injuries and fibrosis in rats. Present results are in accord with previ-ous findings [18, 19].

In the present study, rats treated with CCl4 for 8 weeks exhibited inflammation and fibrosis. Furthermore, serum ALT, AST, ALP, and LDH were significantly increased in CCl4-induced rats. Morphological data further showed the infiltration of inflammatory cells and collagen deposition in the livers after CCl4 treatment. However, every single damage parameter was

and GB treatment. Activated HSCs can upregu-late expression of cytoskeletal proteins, such as α-SMA, a crucial step for cellular differentia-tion. Thus, α-SMA has been considered a mark-er of HSC activation, taken to reflect the degree of liver fibrosis in other studies [24]. In the pres-ent study, α-SMA expression increased in response to CCl4 injections. However, after treat-ment with silymarin or GB, α-SMA protein expression returned to baseline levels, sug-gesting the reversibility of liver fibrosis.

Inflammatory response plays an important role in the progression of liver fibrosis. Pro-inflammatory cytokines, such as TNF-α and IL-1β, have been shown to increase in livers with CCl4-induced hepatitis [25, 26]. Pro-inflammatory cytokines and chemokines stimulate HSCs transformation into myofibroblasts, play-ing a role in activating the production of ECM. Thus, liver fibrosis is closely related to liver inflammation. Results of this study demonstrat-ed the increased expression of both TNF-α and IL-1β in the liver injury group. Conversely, cur-rent findings demonstrated that liver fibrosis and inflammation markers elicited by CCl4 treat-ment were ameliorated by the protective effects of silymarin and GB. This further indi-cated the anti-fibrotic and anti-inflammatory properties of GB. Taken together, results sug-

Figure 5. GB inhibits expression of TNF-α and IL-1β in the liver or serum. (A) mRNA and (B) Protein expression levels of TNF-α and IL-1β, as deter-mined by reverse transcription-quantitative polymerase chain reaction and ELISA. GAPDH served as the internal control. Data are presented as the mean ± standard deviation. #P < 0.05 vs. control group; *P < 0.05 vs. model group.

demonstrably reversed by sily-marin and different doses of GB.

Progression of liver fibrosis is characterized by the accumu-lation of extracellular matrix (ECM) proteins in injured tis-sues. Upon liver chronic injury, hepatic stellate cells (HSCs) are activated by TGF-β1 secret-ed via Kupfer cells [20, 21], allowing trans-differentiation into myofibroblast-like cells, which propagates ECM deposi-tion [22, 23]. FN and HA are key components of the ECM. Their expression levels are closely associated with hepatic fibrosis, cellular damage, dif-ferentiation, and repair. In the present study, FN and HA expression increased after CCl4 treatment. It subsequent-ly decreased with silymarin


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gest that the hepatoprotective capacity of GB, including inhibition of ECM deposition and the resulting histopathology, is probably linked to modulated expression of inflammatory factors.

Acknowledgements

The present study was supported by the Na- tional Natural Science Foundation of China (grant No. 81303125), Nature Science Foun- dation of the Fujian Province of China (No. 2017J01845), and Developmental Fund of Chen Keji Integrative Medicine (CKJ2013015).

Disclosure of conflict of interest

None.

Address correspondence to: Dr. Zhenfeng Hong, Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Qiuyang Road, Shangjie Minhou, Fuzhou 350122, Fujian, China. Tel: +86-591-22861012; Fax: +86-591-22861012; E-mail: [email protected]

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