[Effect of Combination Therapy of Tetramethylpyrazine with Methotrexate on Inflammatory Reac- tions and Hemorheology in Collagen-induced Arthritis Rats].

OBJECTIVE: To explore the effect of combination therapy of tetramethylpyrazine (TMP) with methotrexate (MTX) on collagen induced arthritis (CIA) rats. METHODS: Totally 55 male SD rats were stratified by body weight. Nine of them were randomly recruited as the normal control group. The rest 46 were immunized with type II bovine collagen (C II) for establishing rheumatoid arthritis (RA) model. Forty successfully modeled rats were randomly divided into 4 groups according to swollen toe degree, i.e., the CIA group, the TMP group, the MTX group, and the TMP plus MTX group, 10 in each group. Rats in the MTX group were administered with MTX (1. 2 mg/kg) , once per week for 4 continuous weeks. Those in the TMP group were administered with 40 mg/kg TMP, once per day for 10 continuous days, and then discontinued for 7 successive days, and continued for another 10 successive days. Rats in the TMP plus MTX group were administered with a mixture of equal dose MTX and TMP, and when MTX was discontinue, TMP was administered according to the way in the TMP group. Equal volume of saline solution was given to rats in the normal control group and the CIA group. Clinical parameters including ankle width (mediolateral diameter) and hindpaw swelling were measured at day 0, 4, 11, 18, and 26 after treatment. Rats were sacrificed 28 days after treatment, their knee joints and ankle joints were collected for pathological analyses. Serum levels of IL-1ß, IL-6, and IL-17A were detected by ELISA. Changes of fibrinogen (FIB) and platelet aggregation rate (PAg) were detected. RESULTS: Compared with the normal control group, the ankle width and hindpaw swelling increased significantly (P < 0.01), contents of FIB and PAg increased obviously (P < 0.05, P < 0.01), serum levels of IL-1ß, IL-6, and IL-17 increased remarkably (P <0. 01) in the CIA group. Obvious cell proliferation, inflammatory cell infiltration, hyperemia and edema of synovial tissues could be seen. Pannus formed and immerged in cartilages, resulting in necrosis. Compared with the model group, changes of ankle width and hindpaw swelling were all alleviated in each medicated group (P <0. 05, P <0. 01). Of them, the effect was superior in the MTX group to that of the TMP group and the MTX plus TMP group (P < 0.05, P < 0.01). Contents of FIB, serum levels of IL-1ß and IL-6 decreased significantly in the MTX group (P < 0.05). Contents of FIB, serum levels of IL-1ß and IL-6 decreased significantly in the TMP group and the MTX plus TMP group (P < 0.05). Besides, serum levels of FIB and IL-6 were obviously lower in the MTX plus TMP group than in the TMP group and the MTX group (P < 0.01). Levels of PAg and IL-17A were more significantly lowered in the TMP group than in the MTX plus TMP group and the MTX group. Pathological changes could be alleviated in each medicated group, with the optimal effect obtained in the MTX plus TMP group. CONCLUSION: Combination of TMP with MTX could significantly ameliorate inflammatory reactions and FIB contents of CIA rats.

The effect of JuanBiQiangGu granules in combination with methotrexate on joint inflammation in rheumatoid arthritis: a randomized controlled trial.

Background: Rheumatoid arthritis (RA) joint inflammation severely affects joint function and quality of life in patients and leads to joint deformities and limb disability. The non-steroidal anti-inflammatory drugs used in the treatment of RA do not fully control the progression of joint inflammation and bone destruction and have notable adverse reactions. Traditional Chinese medicine formula JuanBiQiangGu Granules (JBQG) are commonly used for the treatment of RA inflammation and delay of bone destruction, but has not been evaluated through high-quality clinical studies. There is a pressing need for well-designed, randomized, parallel, controlled clinical studies to evaluate the exact effect of JBQG on RA joint inflammation and improvement of patient quality of life. Methods: This is a randomized, parallel, controlled clinical study in which 144 patients with rheumatoid arthritis who met the inclusion criteria were randomly assigned to 2 groups in a 1:1 ratio. The JBQG group received methotrexate 7.5 mg qw and JBQG granules 8 mg tid, while the MTX group received methotrexate 7.5 mg qw. The endpoint was 12 weeks after treatment. Relevant indices at baseline, 4 weeks, 8 weeks, and 12 weeks after treatment were observed and recorded, and DAS28-ESR, HAQ-DI, and Sharp scores were recorded for each patient. Blood samples were collected to test for CRP, ESR, TNF-α, IL-1ß, IL-6, IL-17, and INF-γ, and adverse reactions and liver and kidney function (AST, ALT, Cr, BUN) were recorded for safety assessment. After 12 weeks of treatment, the effect of JBQG granules on disease activity, improvement in bone damage, and patient quality of life scores and safety in RA patients were evaluated. Results: A total of 144 subjects completed treatment (71 in the JBQG group and 73 in the MTX group) and were included in the analysis. At baseline, there were no significant differences between the groups in terms of the observed indicators (p > 0.05). After treatment, 76.06% of patients in the JBQG group had DAS28-ESR levels below or equal to Low, including 45.07% in Remission and 5.63% in High, compared to 53.1% in the MTX group below or equal to Low, 12.33% in Remission, and 17.81% in High. CRP was significantly reduced (8.54 ± 5.87 vs. 11.86 ± 7.92, p < 0.05, p = 0.005), ESR was significantly reduced (15.1 ± 6.11 vs. 21.96 ± 9.19, p < 0.0001), TNF-α was significantly reduced (1.44 ± 0.83 vs. 1.85 ± 1.07, p < 0.05, p = 0.011), IL-17 was significantly reduced (0.53 ± 0.33 vs. 0.71 ± 0.38, p < 0.05, p = 0.004), and INF-γ was significantly reduced (3.2 ± 1.51 vs. 3.89 ± 1.77, p < 0.05, p = 0.014). The median (IQR) OPG in the JBQG group was 2.54 (2.21-3.01), significantly higher than in the MTX group 2.06 (1.81-2.32), p < 0.0001), and the median (IQR) ß-CTX in the JBQG group was 0.4 (0.32-0.43), significantly lower than in the MTX group 0.55 (0.47-0.67), p < 0.0001). The median (IQR) VSA scores were 2 (1-3), a decrease from 3 (2-4) in the MTX group (p < 0.0001). The median (IQR) Sharp scores were 1 (1-2), a decrease from 2 (1-2) in the MTX group, but the difference was not statistically significant (p > 0.05, p = 0.28). The median (IQR) HAQ-DI scores were 11 (8-16), significantly lower than in the MTX group 26 (16-30) (p < 0.0001). The median (IQR) AST in the JBQG group was 16 (12-20), with a significant difference compared to the MTX group 19 (13-25) (p < 0.01, p = 0.004); the median (IQR) ALT in the JBQG group was 14 (10-18), with a significant difference compared to the MTX group 16 (11-22.5) (p < 0.05, p = 0.015). There were no statistically significant differences in Cr or BUN (p > 0.05). Conclusion: JuanBiQiangGu Granules can be used to treat patients with rheumatoid arthritis, alleviate joint inflammation, reduce the incidence of adverse reactions to methotrexate, and has good safety. Clinical Trial Registration: http://www.chinadrugtrials.org.cn/index.html; identifier: ChiCTR2100046373.

Ingredients of Huangqi decoction slow biliary fibrosis progression by inhibiting the activation of the transforming growth factor-beta signaling pathway.

BACKGROUND: Huangqi decoction was first described in Prescriptions of the Bureau of Taiping People's Welfare Pharmacy in Song Dynasty (AD 1078), and it is an effective recipe that is usually used to treat consumptive disease, anorexia, and chronic liver diseases. Transforming growth factor beta 1 (TGFß1) plays a key role in the progression of liver fibrosis, and Huangqi decoction and its ingredients (IHQD) markedly ameliorated hepatic fibrotic lesions induced by ligation of the common bile duct (BDL). However, the mechanism of IHQD on hepatic fibrotic lesions is not yet clear. The purpose of the present study is to elucidate the roles of TGFß1 activation, Smad-signaling pathway, and extracellular signal-regulated kinase (ERK) in the pathogenesis of biliary fibrosis progression and the antifibrotic mechanism of IHQD. METHODS: A liver fibrosis model was induced by ligation of the common bile duct (BDL) in rats. Sham-operation was performed in control rats. The BDL rats were randomly divided into two groups: the BDL group and the IHQD group. IHQD was administrated intragastrically for 4 weeks. At the end of the fifth week after BDL, animals were sacrificed for sampling of blood serum and liver tissue. The effect of IHQD on the TGFß1 signaling pathway was evaluated by western blotting and laser confocal microscopy. RESULTS: Decreased content of hepatic hydroxyproline and improved liver function and histopathology were observed in IHQD rats. Hepatocytes, cholangiocytes, and myofibroblasts in the cholestatic liver injury released TGFß1, and activated TGFß1 receptors can accelerate liver fibrosis. IHQD markedly inhibited the protein expression of TGFß1, TGFß1 receptors, Smad3, and p-ERK1/2 expression with no change of Smad7 expression. CONCLUSION: IHQD exert significant therapeutic effects on BDL-induced fibrosis in rats through inhibition of the activation of TGFß1-Smad3 and TGFß1-ERK1/2 signaling pathways.

[Formula-syndrome correlation study of three classical anti-jaundice formulas in inhibition of liver fibrosis induced by dimethylnitrosamine in rats].

OBJECTIVE: To investigate the effects of three classical anti-jaundice formulas Yinchenhao Decoction (YCHD). Yinchen Wuling San (YCWLS) and Zhizi Baipi Decoction (ZZBPD) on liver fibrosis induced by dimethylnitrosamine (DMN) in rats and explore the formula-syndrome relationship. METHODS: Forty-eight rats were weighted, and divided into five groups: normal control, DMN, YCHD, YCWLS and ZZBPD groups using stratified random method. Liver fibrosis in rats was induced by intraperitoneal injection of DMN for 4 weeks at the dosage 10 mg/kg body weight, once per day for 3 consecutive days in each week. Each groups was gavaged with distilled water (control or model group), YCHD, YCWLS and ZZBPD respectively in the last 2-week modeling period. At the end of the 4th week, rats were sacrificed and their blood sample and liver tissue were taken for detecting pathology and analyzing of hydroxyproline (Hyp) and liver function. RESULTS: Compared with the normal control group, DMN model group showed liver damage and liver fibrosis with remarkably increased Hyp content (P<0.01) and abnormal liver function (P<0.01). Liver fibrosis induced by DMN in rats was improved by administration of YCHD. Compared with DMN model group, content of Hyp was decreased remarkably (P<0.01) and liver function and hepatic histology were improved significantly after 2 weeks of YCHD treatment (P<0.05 or P<0.01)., and content of hepatic Hyp was decreased in the ZZBPD groups. Comparison of various parameters between groups showed that content of serum total bilirubin was decreased significantly in the YCWLS group (P<0.01), and content of hepatic Hyp was decreased in the ZZBPD group. CONCLUSION: Efficacy of YCHD in inhibition of liver fibrosis induced by DMN in rats is far better than YCWLS and ZZBPD. The pathogenesis of the liver fibrosis induced by DMN is mainly damp-heat. In addition, both damp and heat pathogenic factors play very important role in this model. The syndrome is highly correlated with the prescription YCHD.

[Effects of Chinese herbal medicine Yinchenhao Decoction on expressions of apoptosis-related genes in dimethylnitrosamine- or carbon tetrachloride-induced liver cirrhosis in rats].

OBJECTIVE: To investigate the different efficacy of Yinchenhao Decoction (YCHD), a compound traditional Chinese herbal medicine, for liver cirrhosis induced by dimethylnitrosamine (DMN) or carbon tetrachloride (CCl(4)) in rats. METHODS: To induce liver fibrosis, 0.5% DMN solution (2mL/kg body weight, i.p.) was given three consecutive days a week to male Wistar rats for 4 weeks. Cirrhotic rats were randomly divided into DMN group, YCHD group, Xiaochaihu decoction group by the end of the fourth week to accomplish a 2-week recipe treatment course. In CCl(4)-induced liver fibrosis model, 50% CCl(4)-olive solution was injected subcutaneously to rats at a dose of 2 mL/kg body weight twice a week to duplicate rat cirrhosis model. After 8 weeks, rats were divided into CCl(4) group, CCl(4) plus YCHD group and Xiaochaihu decoction group. For the YCHD group, YCHD was administered intragastrically once a day for 4 weeks. For DMN or CCl(4) model, by the end of 6 or 12 weeks respectively, rats were sacrificed for sampling to detect liver function, hepatic histological changes, hydroxyproline (Hyp) content and apoptosis-related gene expressions. RESULTS: In DMN liver fibrosis model, hepatic fibrosis was obvious at week 2 and cirrhosis was evident at week 4 in DMN-treated rats. Compared to 6-week DMN group, hepatic pathological changes and liver function were improved significantly and content of Hyp decreased remarkably in YCHD group. In CCl(4)-induced liver fibrosis model, hepatic fibrosis was obvious at 8 weeks and cirrhosis was evident at 12 weeks in CCl(4)-treated rats. Compared to 12-week CCl(4) group, hepatic pathological changes and liver function were not obviously improvement in YCHD group. The results of gene chip showed that YCHD significantly decreased Fas, Bax and caspase-3 gene expressions, and increased Bcl-xL gene expression in the liver of DMN model. However, in the model induced by CCl(4), YCHD did not inhibit hepatocyte apoptosis induced by CCl(4), but increased tyrosine kinase receptor gene expression by 4.8 times. CONCLUSION: YCHD exerts more significant therapeutic effects on DMN-induced than CCl(4)-induced cirrhosis in rats in Hyp content and pathological change in liver tissue.

Yinchenhao decoction suppresses rat liver fibrosis involved in an apoptosis regulation mechanism based on network pharmacology and transcriptomic analysis.

Yinchenhao decoction (YCHD) is a classical Traditional Chinese Medicine (TCM) formula that has been widely used in the treatment of liver fibrosis caused by chronic hepatitis B and jaundice for more than 1800 years. The purpose of this study was to investigate the apoptosis regulation mechanisms of YCHD and its active components suppresses liver fibrosis. The active components and putative targets of YCHD were predicted by network pharmacology approach. Functional and pathway enrichment analysis were presented in the present study by using clusterProfiler. Further, experimental validation was done by using terminal deoxynucleotidyl transferase (TDT) dUTP nick end labelling (TUNEL) assay and western blotting in dimethylnitrosamine (DMN)-induced liver fibrosis rats, and cell proliferation assay, apoptosis assay, and western blotting in human hepatic L02 cells and LX2 cells. 45 active compounds in YCHD formula, 592 potential target proteins and 1191 liver fibrosis-related human genes were identified. Functional and pathway enrichment analysis indicated that YCHD obviously influenced TNF, PI3K-Akt signaling pathways. Further, In vivo experiment indicated that YCHD treatment not only attenuated the symptoms of liver fibrosis, but also decrease the apoptosis of hepatic parenchyma cells. Moreover, in vitro experiments showed that rhein, kaempferol and quercetin treatments remarkably decreased the protein levels of cleaved caspase-3 and increased p-ERK1/2, PI3K and Bcl-XL protein expression in TNF-α-stimulated L02 cells. On the contrary, rhein, kaempferol, aloe-emodin and quercetin inhibited the proliferation of LX2 cells and up-regulated the protein levels of Bax and cleaved caspase-8. In conclusion, 45 active components and 296 potential targets of YCHD against liver fibrosis were identified by the analysis of network pharmacology and transcriptomics combination. The mechanisms of YCHD against liver fibrosis were involved in the regulation of multiple targets, especially affecting the apoptosis-related signaling pathways.

Urinary metabolomics analysis identifies key biomarkers of different stages of nonalcoholic fatty liver disease.

AIM: To identify a panel of biomarkers that can distinguish between non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), and explore molecular mechanism involved in the process of developing NASH from NAFLD. METHODS: Biomarkers may differ during stages of NAFLD. Urine and blood were obtained from non-diabetic subjects with NAFLD and steatosis, with normal liver function (n = 33), from patients with NASH, with abnormal liver function (n = 45), and from healthy age and sex-matched controls (n = 30). Samples were subjected to metabolomic analysis to identify potential non-invasive biomarkers. Differences in urinary metabolic profiles were analyzed using liquid chromatography tandem mass spectrometry with principal component analysis and partial least squares-discriminate analysis. RESULTS: Compared with NAFLD patients, patients with NASH had abnormal liver function and high serum lipid concentrations. Urinary metabonomics found differences in 31 metabolites between these two groups, including differences in nucleic acids and amino acids. Pathway analysis based on overlapping metabolites showed that pathways of energy and amino acid metabolism, as well as the pentose phosphate pathway, were closely associated with pathological processes in NAFLD and NASH. CONCLUSION: These findings suggested that a panel of biomarkers could distinguish between NAFLD and NASH, and could help to determine the molecular mechanism involved in the process of developing NASH from NAFLD. Urinary biomarkers may be diagnostic in these patients and could be used to assess responses to therapeutic interventions.

Therapeutic mechanism of Yin-Chén-Hao decoction in hepatic diseases.

Yin-Chén-Hao decoction (YCHD) is a traditional Chinese medicine formula composed of capillaris (Artemisia capillaris), gardenia (Gardenia jasminoides), and rhubarb (Rheum rhabarbarum) that is used for the treatment of damp-heat jaundice. In modern clinics, YCHD is mostly used for hepatic diseases. This review summarizes the biological activities of YCHD and its medical applications. The main active compounds of YCHD are chlorogenic acid, rhein, geniposide, emodin, and scoparone. The pharmacological actions of YCHD include inhibition of hepatic steatosis, apoptosis, necrosis, anti-inflammation, and immune regulation. YCHD could be developed as a new therapeutic strategy for the treatment of hepatic diseases.

Yin-Chen-Hao-Tang alleviates biliary obstructive cirrhosis in rats by inhibiting biliary epithelial cell proliferation and activation.

BACKGROUND: Yin-Chen-Hao-Tang (YCHT) consists of three aqueous extracts from Artemisia capillaris, Gardenia sp., and prepared Rheum rhabarbarum (rhubarb) (3:2:1). YCHT is characterized by its anti-inflammatory properties in liver regulation and relief of jaundice. We aimed to study the effects and mechanisms of action of YCHT on biliary obstructive cirrhosis. MATERIALS AND METHODS: Secondary biliary fibrosis was induced in rats by bile duct ligation (BDL) and scission. One week after BDL, rats were randomly divided into a saline-treated BDL or YCHT-treated BDL group for 4 weeks. Liver function and hepatic hydroxyproline (Hyp) content were assessed. Types I and IV collagen (Col-IV), laminin, fibronectin, alpha smooth muscle actin (α-SMA), and proliferating cell nuclear antigen protein and messenger ribonucleic acid (mRNA) expression were assessed with immunohistochemistry and real-time polymerase chain reaction. RESULTS: In the YCHT-treated BDL group, serum total bilirubin, total bile acids, aspartate aminotransferase, alanine aminotransferase, and γ-glutamyl transferase were lower than those in the sham-operated BDL group. The proliferation of bile ducts in hepatic tissues and the Hyp content and Col deposition were also significantly lower than those in control rats. In addition, α-SMA and Col-IV staining was less obvious, and mRNA expression of Procol-α1 (IV), platelet derived growth factor subunit B (PDGF)-B, connective tissue growth factor, and transforming growth factor-beta in proliferative biliary epithelial cells (BECs) in the YCHT-treated BDL group was significantly lower than those in controls. CONCLUSIONS: YCHT effectively reduces the formation of biliary obstructive cirrhosis mainly via inhibition of BEC proliferation by down-regulation of PDGF-B mRNA expression, inhibition of BEC profibrogenic paracrines, and the epithelial-mesenchymal transition pathological process.

Xiayuxue Decoction ([symbols; see text]) attenuates hepatic stellate cell activation and sinusoidal endothelium defenestration in CCl4-induced fibrotic liver of mice.

OBJECTIVE: To investigate the effects of ancient Chinese medical formula Xiayuxue Decoction ([symbols; see text], XYXD) on activation of hepatic stellate cells (HSCs) and defenestration of sinusoidal endothelial cells (SECs) in CCl4-induced fibrotic liver of mice. METHODS: High performance liquid chromatography was used to identify the main components of XYXD and control the quality of extraction. C57BL/6 mice were induced liver fibrosis by CCl4 exposure and administered with XYXD for 6 weeks simultaneously. Liver tissue was investigated by hematoxylin-eosin and Sirius-red staining. Sinusoidal fenestrations were observed by scanning electronic microscopy and fluorescent immunohistochemistry of PECAM-1 (CD31). Whole liver lysates were detected of α-smooth muscle actin (α-SMA) and type-I collagen by Western blot. Primary rat HSCs-T6 cells were analyzed by detecting α-SMA, F-actin, DNA fragmentation through confocal microscopy, Western blot, terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay and cellomics arrayscan, respectively. RESULTS: Amygdalin and emodin in XYXD were identified. XYXD (993 mg/kg) inhibited Sirius red positive area up to 70.1% (P<0.01), as well as protein levels of α-SMA and type-I collagen by 42.0% and 18.5% (P<0.05) respectively. In vitro, XYXD (12.5 µg/mL, 50 µg/mL) suppressed the activation of HSCs and reversed the myofibroblastic HSCs into quiescent, demonstrated as inhibition of fluorescent F-actin by 32.3% and 46.6% (P<0.05). Besides, XYXD induced the apoptosis of HSC-T6 cells by 20.0% (P<0.05) and 49.5% (P<0.01), evidenced by enhanced TUNEL positivity. Moreover, ultrastructural observation suggested XYXD inhibited defenestration of SECs, which was confirmed by 31.1% reduction of protein level of CD31 (P<0.05). CONCLUSIONS: XYXD inhibited both HSCs activation and SECs defenestration which accompany chronic liver injuries. These data may help to understand the underlying mechanisms of XYXD for prevetion of chronic liver diseases.