Melatonin Ameliorates Liver Fibrosis Induced by Carbon Tetrachloride in Rats via Inhibiting TGF-β1/Smad Signaling Pathway

Melatonin has been reported to inhibit hepatic fibrosis and the mechanism may be correlated to its anti-oxidant effect. Nevertheless, the mechanism is not completely identified. This study was conducted to investigate the effects of melatonin on TGF-β1/Smad signaling pathway in liver fibrosis in rats. The liver fibrosis model was made by the subcutaneous injection of CC14. The liver pathology changes were detected using hematoxylin and eosin （H＆E） staining and Van Gieson （VG） staining. Serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） activities were measured with an autoanalyzer. Glutathione peroxidase （GPx） activities and levels of malondialdehyde （MDA） and hydroxyproline （Hyp） in liver were evaluated by spectrophotometry. Expression levels of TGF-β1, Smad2/3, phosphorylated Smad2/3 （p-Smad2/3） and Smad7 in liver were detected by immunohistochemistry and Western blot analysis. Results showed that melatonin suppressed CC14-induced liver fibrosis, along with an improvement in histological changes, significant decreases in pathologic grading sores and obvious decreases in Hyp levels in liver. Melatonin improved liver function indicated by decreased serum ALT and AST activities. In addition, melatonin exerted its anti-oxidant effects, as supported by decreased MDA levels and increased GPx activities in liver. Furthermore, melatonin inhibited TGF-β1/Smad pathway, as evidenced by decreased TGF-β1, Smad2/3 and p-Smad2/3 expression and increased Smad7 expression in liver. In conclusion, melatonin may suppress CCl4-induced hepatic fibrosis in rats via inhibiting TGF-β1/Smad pathway. It is possible for melatonin to be a potential reagent to treat and cure liver fibrosis.

Correlation of serum TGF-β1 content with liver fibrosis and Th1/Th2 immune levels in patients with chronic hepatitis b

Objective:To investigate the correlation of serum TGF-β1 content with liver fibrosis and Th1/Th2 immune levels in patients with chronic hepatitis b.Methods: A total of 178 patients who were diagnosed with chronic hepatitis b in our hospital between February 2015 and August 2017 were selected as chronic hepatitis b group, and 100 healthy volunteers who received physical examination in our hospital during the same period were selected as normal control group. The differences in serum levels of TGF-β1, liver fibrosis indexes and Th1/Th2 cytokines were compared between the two groups, and the Pearson test was adopted to evaluate the correlation between serum TGF-β1 content and disease severity in patients with chronic hepatitis b.Results: Serum TGF-β1 level of chronic hepatitis b group was higher than that of normal control group;serum liver fibrosis indexes CⅣ, PC-Ⅲ, HA and LN levels were higher than those of normal control group;serum Th1 cytokines IFN-γ and IL-12 levels were lower than those of normal control group whereas Th2 cytokines IL-4 and IL-13 levels were higher than those of normal control group. Pearson test showed that the serum TGF-β1 level in patients with chronic hepatitis b was positively correlated with the degree of liver fibrosis and Th1/Th2 immune imbalance.Conclusions: Serum TGF-β1 expression is abnormally high in patients with chronic hepatitis b and directly correlated with the degree of liver fibrosis and immune imbalance.

Effect of All-trans Retinoic Acid on Liver Fibrosis Induced by Common Bile Duct Ligation in Rats

The aim of this study was to investigate the effect and possible mechanism of all-trans retinoic acid （ATRA） on liver fibrosis induced by common bile duct ligation （CBDL） in rats. Fifty-three female Wistar rats were randomly divided into 5 groups： sham operation group （group J, 5 animals） and groups A, B, C and D （12 animals in each group）. The rats in groups A, B, C and D were subjected to CBDL to induce liver fibrosis, while those in group J to sham operation. From the 3rd week the rats in groups B, C and D respectively received daily administration of ATRA via gas- tric tube at three different doses [0.1, 1.5 and 7.5 mg/kg body weight （BW）]. Animals were sacrificed at 6th week. Rats＇ liver tissues were observed for pathologic changes under a light microscope. The protein levels of type Ⅰ collagen （COLⅠ）, matrix metalloproteinase-2 （MMP2）, MMP13 and tissue inhibitors of metalloproteinase-1 （TIMP-1） in liver tissues were determined by immunohistochemical techniques. The expression levels of TGF-β1 and CTGF mRNA in liver tissues were detected by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR）. The results showed that loss of normal hepatic architecture and formation of obvious fibrosis were observed in group A, while ATRA treatment for 4 weeks notably alleviated the pathological changes of hepatocytes. The expres- sion of COL Ⅰ and TIMP-1 proteins in group A was increased, while decreased in ATRA-treated CBDL groups （P〈0.05）. ATRA （1.5 and 7.5 mg/kg BW） reduced the expression levels of COLⅠ protein more greatly than that of 0.1 mg/kg BW （P〈0.05）. ATRA treatment increased the protein levels of MMP2 and MMP13. The expression levels of TGF-β1 and CTGF mRNA in group A were increased. In comparison with group A, the mRNA levels of TGF-β1 and CTGF in ATRA-treated CBDL groups were significantly decreased （P〈0.05）. It was concluded that ATRA could inhibit CBDL-induced liver fibrosis in rats by suppressing the expression of TGF-β1 and CTGF so as to di- minish the inhibition of TIMP-1 on MMP2 and MMP13 and increase the activity of MMP2 and MMP13.

Effect of Anluohuaxian Tablet Combined with γ-IFN on Schistosomal Liver Fibrosis

The therapeutic effects of anluohuaxian tablet combined with γ-IFN on schistosomal liver fibrosis and its mechanism were studied in a murine model and clinical cases of schistosomal liver fibrosis, Fifty Kunming mice were randomly divided into 5 groups： normal control group, infection control group, anluohuaxian tablet-treated group, γ-IFN-treated group and combined treatment （anluohuaian tablet＋γ-IFN） group. Pathologic changes in liver, including hepatic pigmentation and the size of schistosomal egg granuloma, were observed by HE staining after treatment for 8 weeks. The expression of the type Ⅰ and Ⅲ collagen, and TIMP-1 was detected by immunohistochemistry. TGF-β1 mRNA expression was examined by real-time fluorescent quantitative PCR. Sixty patients with schistosomal liver fibrosis were divided into treatment group and control group. The patients in treatment group were treated with anluohuaxian tablet in combination with γ-IFN for 6 months. Before and after treatment, the changes of symptoms and signs, liver function, serum liver fibrosis indexes and imaging indexes were observed. The results showed that as compared with infection control group, all forms of treatments relieved the hepatic pathological injury with apparently diminished size of schistosomal egg nodules and decreased percentage of pigmentation （P〈0.05）. Furthermore, the expression of collagen Ⅰ and Ⅲ, TIMP-1, and TGF-β1 mRNA in combined treatment group was significantly decreased as compared with anluohuaxian tablet-treated and γ-IFN-treated groups （P〈0.05）. In the clinical observation, the serum liver fibrosis indexes, the portal vein width as well as the spleen thickness was significantly reduced in treatment group as compared with control group （P〈0.05）. It was concluded that the combined use of anluohuaxian tablet with γ-IFN in schistosomal liver fibrosis could protect liver function, alleviate liver fibrosis, and could be used as a choice in treating patients with schiatosomal liver fibrosis.

Upregulation of GLT25D1 in Hepatic Stellate Cells Promotes Liver Fibrosis via the TGF-β1/SMAD3 Pathway In Vivo and In vitro

Background and Aims:Collagenβ(1-O)galactosyltransferase 25 domain 1(GLT25D1)is associated with collagen production and glycosylation,and its knockout in mice results in embryonic death.However,its role in liver fibrosis remains elusive,particularly in hepatic stellate cells(HSCs),the primary collagen-producing cells associated with liver fibrogenesis.Herein,we aimed to elucidate the role of GLT25D1 in HSCs.Methods:Bile duct ligation(BDL)-induced mouse liver fibrosis models,primary mouse HSCs(mHSCs),and transforming growth factor beta 1(TGF-β1)-stimulated LX-2 human hepatic stellate cells were used in in vivo and in vitro studies.Stable LX-2 cell lines with either GLT25D1 overexpression or knockdown were established using lentiviral transfection.RNA-seq was performed to investigate the genomic differences.HPLCMS/MS were used to identify glycosylation sites.Scanning electronic microscopy(SEM)and second-harmonic generation/two-photon excited fluorescence(SHG/TPEF)were used to image collagen fibril morphology.Results:GLT25D1 expression was upregulated in nonparenchymal cells in human cirrhotic liver tissues.Meanwhile,its knockdown attenuated collagen deposition in BDL-induced mouse liver fibrosis and inhibited mHSC activation.GLT25D1 was overexpressed in activated versus quiescence LX-2 cells and regulated in vitro LX-2 cell activation,including proliferation,contraction,and migration.GLT25D1 also significantly increased liver fibrogenic gene and protein expression.GLT25D1 upregulation promoted HSC activation and enhanced collagen expression through the TGF-β1/SMAD signaling pathway.Mass spectrometry showed that GLT25D1 regulated the glycosylation of collagen in HSCs,affecting the diameter of collagen fibers.Conclusions:Collectively,the upregulation of GLT25D1 in HSCs promoted the progression of liver fibrosis by affecting HSCs activation and collagen stability.

Effects of novel Fufang Biejia Ruangan Tablets with sheep placenta as substitute for Hominis Placenta on CCl4-induced liver fibrosis

Objective:Fufang Biejia Ruangan Tablet(FBRT) is widely used for the treatment of liver fibrosis.However,Hominis Placenta(HP),as an important adjuvant of FBRT,has been restricted for medicinal using due to the limited availability,ethical controversy and safety issues.The present study aimed to investigate the therapeutic effects of novel FBRT(N-FBRT) with sheep placenta(SP) as substitute for HP on liver fibrosis and explore its possible mechanisms.Different dosages of SP in N-FBRT were also evaluated.Methods:Rats were subcutaneously injected with CCl_(4)to induce liver fibrosis and then treated with NFBRT and FBRT.The anti-hepatic fibrosis effect was determined based on biomarkers analysis of liver function and hepatic fibrosis,and the liver pathology was visualized by H&E staining and Masson staining.The oxidative stress and inflammatory cytokines were also detected.Immunohistochemical staining of a-SMA,real time PCR and Western blotting were performed to evaluate hepatic stellate cells(HSCs)activation and TGF-β1/Smad signaling pathway.Results:N-FBRT and FBRT could ameliorate CCl_(4)-induced liver fibrosis and improve liver function,as evidenced by lowering serum biomarkers levels of liver function and hepatic fibrosis,and decreasing hepatic Hyp content and collagen deposition,and improving the hepatic morphology and architecture changes.Moreover,the anti-liver fibrosis effect was better when the dosage of SP used in N-FBRT was 1/2 of HP in FBRT.Administration of N-FBRT markedly alleviated oxidative stress and inflammatory cytokines,and inhibited a-SMA expression.Furthermore,the mRNA expression of Col Ⅰ,Col Ⅲ,a-SMA and TGF-β1,and proteins expression of a-SMA,TGF-β1,Smad2/3 and p-Smad2/3 were significantly down-regulated by N-FBRT treatment.Conclusion:SP can be used as substitute for HP to prepare N-FBRT for the treatment of liver fibrosis and the anti-liver fibrosis effect of N-FBRT is achieved by eliminating oxidative stress and inflammation,and inhibiting HSCs activation and ECM production by blocking TGF-β1/Smad signaling pathway.