Lian-Jun Xing

Berberine reducing insulin resistance by up-regulating IRS-2 mRNA expression in nonalcoholic fatty liver disease (NAFLD) rat liver

This study was performed to investigate the molecular mechanism and the therapeutic effect of berberine on nonalcoholic fatty liver disease (NAFLD). Rat models were given a high-fat diet (42% kcal) until they developed NAFLD, then were given normal saline (n=10), berberine (n-=10) at 187.5mg/kg/day, or pioglitazone (n=10) at 10.0mg/kg/day intragastrically for 4 weeks, respectively, and evaluated by hyperinsulinemic euglycemic clamping for insulin sensitivity. Serum biochemical markers and liver triglyceride (TG) were analyzed, real-time RT-PCR for mRNA expression and western blotting for protein expression of insulin receptor (IR) and insulin receptor substrate-2 (IRS-2) in liver tissues were performed, and hepatic histopathology in the rat models with NAFLD at the end of treatment was compared with normal controls (n=10). The NAFLD rats developed insulin resistance, showing increased fasting blood glucose and insulin levels, decreased glucose infusion rate, increased weight of epididymal fat (g/100g body weight), obvious hepatic steatosis and inflammation, and down-regulated IRS-2 mRNA and protein levels compared with normal controls (all P<0.05). In comparison with those treated with saline, model rats treated with berberine or pioglitazone underwent significant recovery, including up-regulated IRS-2 mRNA and protein (all P<0.05). Our results indicate that berberine may improve insulin resistance of NAFLD by up-regulating mRNA and protein levels of IRS-2, a key molecule in the insulin signaling pathway, suggesting that berberine may be used to treat NAFLD.

Qinggan Huoxue Recipe suppresses epithelial-to-mesenchymal transition in alcoholic liver fibrosis through TGF-β1/Smad signaling pathway.

AIM To investigate the mechanism by which Qinggan Huoxue Recipe (QGHXR) inhibits epithelial-to-mesenchymal transition (EMT) in rats with alcoholic liver fibrosis (ALF). METHODS A total of 75 male SD rats were used to induce ALF. Serum biochemical indicators, including alanine aminotransferase, aspartate aminotransferase, laminin and hyaluronidase, were measured. Liver histopathological changes were evaluated using hematoxylin-eosin and Sirius red staining. EMT was examined by analyzing the expression of the epithelial marker E-cadherin and the mesenchymal markers vimentin and fibronectin using RT-PCR and Western blot. The inhibitory effect of QGHXR on EMT markers, as well as its effect on molecules associated with the transforming growth factor (TGF)-β1/Smad signaling pathway, including TGF-β1, Smad3, snail, occludin, ZO-1 and claudin, was also examined. RESULTS Compared with normal control rats, ALF rats exhibited a decrease in E-cadherin levels (mRNA: ALF 0.16 ± 0.05 vs control 1.00 ± 0.08; protein: ALF 0.09 ± 0.05 vs control 0.70 ± 0.17, P < 0.01) and an increase in vimentin and fibronectin levels (mRNA: 11.43 ± 0.39 vs 1.00 ± 0.19 and 9.91 ± 0.34 vs 1.00 ± 0.44, respectively, P < 0.01; protein: 1.13 ± 0.42 vs 0.09 ± 0.03 and 1.16 ± 0.43 vs 0.09 ± 0.00, respectively, P < 0.01). This indicates that EMT occurred in ALF rats. In addition, the TGF-β1/Smad signaling pathway was activated in ALF rats, as evidenced by the increase in TGF-β1 and snail levels (mRNA: 1.76 ± 0.12 vs 1.00 ± 0.05 and 6.98 ± 0.41 vs 1.00 ± 0.10, respectively, P < 0.01; protein: 1.43 ± 0.05 vs 0.12 ± 0.03 and 1.07 ± 0.29 vs 0.07 ± 0.02, respectively, P < 0.01) and the decrease in Smad3 levels (mRNA: 0.05 ± 0.01 vs 1.00 ± 0.12, P < 0.01; protein: 0.06 ± 0.05 vs 0.89 ± 0.12, P < 0.01). Furthermore, levels of the tight junction markers occludin, ZO-1 and claudin decreased in ALF rats compared with healthy control rats (mRNA: 0.60 ± 0.09 vs 1.00 ± 0.12, 0.11 ± 0.00 vs 1.00 ± 0.12 and 0.60 ± 0.01 vs 1.00 ± 0.08, respectively, P < 0.01; protein: 0.05 ± 0.01 vs 0.87 ± 0.40, 0.09 ± 0.05 vs 0.89 ± 0.18 and 0.04 ± 0.03 vs 0.95 ± 0.21, respectively, P < 0.01). In ALF rats treated with QGHXR, E-cadherin levels increased (mRNA: QGHXR 0.67 ± 0.04 vs ALF model 0.16 ± 0.05, P < 0.01; protein: QGHXR 0.66 ± 0.21 vs ALF model 0.09 ± 0.05, P < 0.01), and vimentin and fibronectin levels decreased (mRNA: 6.57 ± 1.05 vs 11.43 ± 0.39 and 1.45 ± 1.51 vs 9.91 ± 0.34, respectively, P < 0.01; protein: 0.09 ± 0.03 vs 1.13 ± 0.42 and 0.10 ± 0.01 vs 1.16 ± 0.43, respectively, P < 0.01). In addition, QGHXR inhibited the expression of TGF-β1 and increased the expression of Smad3 (mRNA: 1.03 ± 0.11 vs 1.76 ± 0.12, 0.70 ± 0.10 vs 0.05 ± 0.01, respectively, P < 0.05 and P < 0.01; protein: 0.12 ± 0.03 vs 1.43 ± 0.05 and 0.88 ± 0.20 vs 0.06 ± 0.05, respectively, P < 0.01). QGHXR treatment also reduced the levels of the EMT-inducing transcription factor snail (mRNA: 2.28 ± 0.33 vs 6.98 ± 0.41, P < 0.01; protein: 0.08 ± 0.02 vs 1.07 ± 0.29, P < 0.01) and increased the occludin, ZO-1 and claudin levels (mRNA: 0.73 ± 0.05 vs 0.60 ± 0.09, 0.57 ± 0.04 vs 0.11 ± 0.00 and 0.68 ± 0.03 vs 0.60 ± 0.01, respectively, P < 0.01, P < 0.01 and P < 0.05; protein: 0.92 ± 0.50 vs 0.05 ± 0.01, 0.94 ± 0.22 vs 0.09 ± 0.05 and 0.94 ± 0.29 vs 0.04 ± 0.03, respectively, P < 0.01). The effects of QGR and HXR on the TGF-β1/Smad signaling pathway were similar to that of QGHXR; however, the QGR- and HXR-induced changes in vimentin mRNA levels, the QGR-induced changes in fibronectin mRNA levels and the HXR-induced changes in snail and TGF-β1 mRNA levels were not significant. CONCLUSION Qinggan Huoxue Recipe inhibits EMT in ALF rats by modulating the TGF-β1/Smad signaling pathway, suggesting that the mechanism underlying the amelioration of ALF induced by QGHXR is associated with this pathway.

Inhibition of LXRα/SREBP-1c-Mediated Hepatic Steatosis by Jiang-Zhi Granule

Nonalcoholic fatty liver (NAFL) is increasingly recognized as one of the most common causes of chronic liver disease worldwide. Traditional Chinese medicine (TCM), as the alternative and complementary medicine, may provide some profound health benefit. “Jiang-Zhi” Granule (JZG) was composed based on TCM pathogenesis of NAFL: the retention of inner dampness, heat and blood stasis. This study investigated effects of JZG on liver X receptor-α (LXRα)/sterol regulatory element binding protein-1c (SREBP-1c) pathway in high-fat-diet-(HFD-)induced hepatic steatosis, as well as in free-fatty-acid-(FFA-)and T0901317-treated HepG2 cells. The results showed that JZG had an antisteatotic effect on HFD-fed rats. JZG decreased the activation of SREBP-1c through inhibiting LXRα-mediated SREBP-1c transcription, as well as through inhibiting the maturation of SREBP-1c independent of LXRα. These findings may provide molecular evidence for the use of JZG as a promising therapeutic option for NAFL and support us to continue JZG treatment in NAFL. For JZG treatment to be widely accepted, a randomized, double-blind, multicenter, placebo-controlled, phase III trial is ongoing.

[Effects of puerarin on expressions of leptin receptor mRNA, phosphorylated Janus kinase 2/phosphorylated signal transducers and activators of transcription 3 proteins in the liver of rats with non-alcoholic fatty liver].

OBJECTIVE To observe the effects of puerarin on the expressions of leptin receptor mRNA and phosphorylated Janus kinase 2 / phosphorylated signal transducers and activators of transcription 3 (P-JAK2/P-STAT3) proteins in the liver of rats with non-alcoholic fatty liver (NAFLD). METHODS A rat model of NAFLD was successfully established by feeding high-fat diet. All SD rats were randomly divided into blank control group, untreated group, simvastatin-treated group and puerarin-treated group. After four-week treatment, the levels of hepatic triglyceride and total cholesterol were analyzed by using an automatic biochemical analyzer. The pathology of the liver tissue was observed by light microscopy. Serum leptin level was detected by enzyme-linked immunosorbent assay, and the expressions of leptin receptor mRNA and P-JAK2/P-STAT3 proteins in the liver of NAFLD rats were quantified by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis respectively. RESULTS Puerarin significantly decreased the levels of hepatic triglyceride and total cholesterol in NAFLD rats. Fat degeneration and inflammatory reaction in liver tissues of NAFLD rats were ameliorated after puerarin treatment. The serum leptin level was increased and the expressions of leptin receptor mRNA and P-JAK2/P-STAT3 proteins were up-regulated in puerarin-treated group. CONCLUSION Puerarin can effectively attenuate liver lipid disorder and inflammation by improving the leptin resistance and enhancing the expressions of leptin receptor mRNA and P-JAK2/P-STAT3 proteins.

The Functional Difference of Dendritic Cells in HBeAg Negative Chronic Hepatitis B Patients with Three Different Spleen Deficiency Syndromes and the Therapeutic Evaluation of Chinese Medicine Intervention Based on Syndrome Differentiation

Objective. To investigate the dendritic cells (DCs) maturity differences of HBeAg negative chronic hepatitis B (CHB) patients with different spleen deficiency (SD) syndromes and explore the role of syndrome differentiation in the therapeutic evaluation of Chinese medicine. Methods. 120 participants were recruited including three treatment groups in different SD syndrome categories as spleen deficiency with liver depression (SDLD), spleen deficiency with damp heat (SDDH), and spleen deficiency with kidney deficiency (SDKD) and one healthy control group; each group had 30 participants. Corresponding drugs were applied. The outcome measures included DC phenotype, liver function, IL-10, IL-12, and HBV-DNA levels. Results. The surface markers of mature DCs and cytokines levels were different in each group; the positive rate of CD80, CD1a, HLA-DR, and CD1a was the lowest in SDKD group. After 3-month intervention, the expression of CD80, CD86, CD1a, HLA-DR, and IL-12 significantly increased, while ALT, AST, and IL-10 significantly decreased (P < 0.05) in treatment groups. HBV-DNA level also significantly reduced in both SDKD and SDLD groups (P < 0.05). Conclusions. HBeAg negative patients had DCs dysmaturity, and there were differences between different SD syndromes. Chinese medicine intervention according to syndrome differentiation could advance the maturity and function of DCs and improve the therapeutic effect.

Chinese medicinal formula, Qinggan Huoxue Recipe protects rats from alcoholic liver disease via the lipopolysaccharide‑Kupffer cell signal conduction pathway

The Chinese medicinal formula, Qinggan (QG) Huoxue (HX) Recipe (R) exerts a range of pharmacological effects, including reversible steatosis, decreased levels of inflammatory cytokines and lipid peroxidation resistance. The aim of the present study was to determine the specific mechanisms of QGHXR hepatoprotection through the lipopolysaccharide-Kupffer cell (LPS-KC) signal conduction pathway in rats with alcoholic liver disease (ALD). ALD rats were exposed to the compound factors, QGR and HXR. Hematoxylin and eosin staining was conducted to evaluate the pathological changes in the liver following QGHXR treatment and an enzyme-linked immunosorbent assay was performed to measure the content of tumor necrosis factor (TNF)-α in the plasma. Immunohistochemical staining was conducted to examine the expression of cell differentiation antigen (CD) 68 and 14. In addition, western blot analysis and reverse transcription-polymerase chain reaction were used to measure the expression of Toll-like receptor 4 (TLR4), phosphorylated-extracellular regulated protein kinases (p-ERK), nuclear factor (NF)-κB, CD14 and TNF-α. Following stimulation with the compound factors, the rats exhibited increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, as well as marked pathological changes. Furthermore, the related molecules in the LPS-KC pathway were upregulated and QGHXR was identified to be effective in the LPS-KC signal conduction pathway in the ALD rats. QGHXR was superior to QGR and HXR in reducing the serum ALT and AST levels, regulating CD14, TLR4, NF-κB, ERK and TNF-α as well as improving the pathological changes. The results indicated that QGHXR therapy may provide a novel strategy for treating ALD via regulation of the related molecules in the LPS-KC signaling pathway.

Baicalin and puerarin reverse epithelial‑mesenchymal transition via the TGF‑β1/Smad3 pathway in vitro

Epithelial-mesenchymal transition (EMT) occurs in the development of fibrosis and carcinogenesis. EMT is associated with chronic liver injury. Evidence shows that hepatocytes undergo EMT in the adult liver. The Qinggan Huoxue Recipe (QGHXR), a Traditional Chinese Medicinal formula, shows a range of pharmacological effects in treating alcoholic liver disease. The present study aimed to investigate the effect of four major components of QGHXR, baicalin, salvianic acid, puerarin and saikosaponin, on EMT in vitro, and to elucidate the potential mechanism of QGHXR against EMT via the transforming growth factor-β1 (TGF-β1)/Smads signaling pathway. EMT models were established using LO2 hepatocytes and HepG2 cells treated with acetaldehyde in vitro. Acetaldehyde presented a mesenchymal cell characteristic in hepatocytes, accompanied by an increased expression of mesenchymal markers, including vimentin and fibronectin, and decreased E-cadherin. Baicalin and puerarin abrogated the increased expression of vimentin and fibronectin, and rescued E-cadherin expression in acetaldehyde-treated hepatocytes. It was further demonstrated that baicalin and puerarin reduced the gene expression of snail, TGF-β1 and Smad3. A decreased expression of tight function markers, including ZO-1, occludin and claudin, were also found in the acetaldehyde-treated hepatocytes. Barcacin regulated the mRNA level of TGF-βl and snail, and then suppressed the EMT process. This was accompanied by an increased mRNA level of E-cadherin and decreased levels of vimentin and fibronectin, but no significant differences in of Smad3, occludin, ZO-1 and claudin were observed. Puerarin regulated the mRNA level of TGF-βl, Smad3 and snail, suppresing the EMT process, which was accompanied by an increased mRNA level of E-cadherin and decreased levels of vimentin and fibronectin, along with increased levels of occludin, ZO-1 and claudin. When the snail gene was silent, barcacin and puerarin did not show significant effects in the acetaldehyde-treated cells. The results presented a novel mechanism through which baicalin and puerarin modulated hepatocyte EMT to improve liver fibrosis.

Relationship between the phenotypes and functions of peripheral blood dendritic cells and the different spleen deficiency syndrome types in patients with chronic hepatitis B

OBJECTIVE To study the phenotypes and functions of dendritic cells (DCs) derived from peripheral blood monocytes of chronic hepatitis B (CHB) patients with different traditional Chinese medicine (TCM) syndrome types, and to explore the relationship between TCM syndrome type and DC functions. METHODS Sixty CHB patients were included in this study. All the CHB patients were divided into spleen deficiency and liver stagnation, spleen deficiency and dampness-heat and deficiency of both spleen and kidney groups according to TCM syndrome diagnosis standard. There were 20 cases in each group, and ten healthy people were included as normal control. The volunteers peripheral blood was collected for monocyte separation, biochemical test and hepatitis B virus DNA loads detection. DCs were induced and isolated from peripheral blood monocytes, and then the expressions of surface markers CD80, CD86, CD1a and HLA-DR were detected by flow cytometric analysis method. Interleukin-10 (IL-10) production of the DCs was quantified by enzyme-linked immunosorbent assay. RESULTS The proliferation of DCs in the CHB patients was slower than that in the healthy volunteers (P<0.05). The expressions of DC surface molecules such as CD80, CD86, and CD1a were obviously decreased in the CHB patients as compared with those in the healthy volunteers (P<0.05). More over, expressions of DC surface molecules were different among CHB patients with different TCM syndrome types. The positive expressions of CD80, CD1a, and HLA-DR in the CHB patients with spleen deficiency and liver stagnation were obviously higher than those in the CHB patients with deficiency of both spleen and kidney (P<0.05), and the CD1a expression in the CHB patients with spleen deficiency and dampness-heat was higher than that in the CHB patients with deficiency of both spleen and kidney (P<0.05). In DC culture supernatant, the IL-10 concentration of the CHB patients with deficiency of both spleen and kidney was higher than that of the CHB patients with spleen deficiency and liver stagnation (P<0.05), and the IL-10 concentrations of the CHB patients with different TCM syndrome types were higher than that of the healthy volunteers (P<0.05). CONCLUSION During the pathogenic course of CHB, the phenotypes and functions of DCs are different in CHB patients with different TCM syndrome types. It suggests that there is a correlation between TCM syndrome type and body immunity function.