Pure total flavonoids from citrus attenuate non-alcoholic steatohepatitis via regulating the gut microbiota and bile acid metabolism in mice.

BACKGROUND: Our previous studies found that Pure total flavnoids from citrus (PTFC) can effectively improve non-alcoholic steatohepatitis (NASH) in mice. Here, we discuss on the mechanism of PTFC in treating NASH with focus on the regulation of the gut microbiota and bile acid metabolism. METHODS: C57BL/6 J mice were randomly divided into three groups: normal diet group (Normal), high-fat diet group (HFD) and high-fat + PTFC treatment group (PTFC). Mice in the Normal group were fed chow diet, while the other groups were fed high fat diet (HFD) for 16 weeks. In the 5th week, the mice in the PTFC group were treated with 50 mg/kg/day PTFC for an additional twelve weeks. After sacrifice, histopathology of the liver was assessed, and the gut microbial composition was analyzed by 16S rDNA gene sequencing. Bile Acid profiles in serum were determined by ultraperformance liquid chromatography (UPLC-MS/MS). RESULTS: PTFC intervention significantly attenuated HFD-induced NASH symptoms compared with the HFD group in mice. 16S rDNA sequencing showed that PTFC treatment increased the phylogenetic diversity of the HFD-induced microbiota dysbiosis. PTFC intervention significantly increased the relative abundances of Bacteroidaceae and Christensenellaceae. Furthermore, PTFC reduced the content of toxic bile acids, such as TDCA, DCA, TCA, CA and increased the ratio of secondary to primary bile acids. FXR and TGR5 deficiency were significantly alleviated. CONCLUSION: PTFC can improve NASH via the the gut microbiota and bile acid metabolism.

Berberine attenuates non-alcoholic steatohepatitis by regulating chemerin/CMKLR1 signalling pathway and Treg/Th17 ratio.

To observe the therapeutic effect of berberine (BBR) on non-alcoholic steatohepatitis (NASH) in rats and the underlying mechanism. A rat model of NASH was established by a high-fat diet, and BBR was used as treatment. Haematoxylin-eosin staining and Oil Red O staining were used to observe the pathological changes in the liver tissue. Western blotting and real-time PCR were used to measure the mRNA and protein levels in the liver. Flow cytometry was performed to detect the number of intrahepatic lymphocyte subtypes. The expression of pro-inflammatory cytokines in the peripheral blood was measured by ELISA. An automatic biochemical method was used to examine the level of blood lipids in the blood. Compared with the rats in the model group, the rats in the BBR group showed significantly improved liver histopathology and serum pro-inflammatory cytokines and free fatty acid (FFA) levels. Moreover, the protein and mRNA expression of chemerin, CMKLR1 and CCR2 in the liver were obviously reduced by BBR treatment. In addition, the high-fat diet remarkably reduced the intrahepatic Treg/Th17 ratio, which could be recovered by BBR treatment. Berberine can ameliorate non-alcoholic steatohepatitis, and its mechanism may be related to restoring the Treg/Th17 ratio, regulating the chemerin/CMKLR1 signalling pathway to reduce liver inflammation and reducing lipid deposition.

Efficacy of Banxia Xiexin decoction in a rat model of chronic atrophic gastritis.

OBJECTIVE: To investigate the effectiveness of Banxia Xiexin decoction (BXD) in a rat model of chronic atrophic gastritis (CAG). METHODS: Sixty 6-week-old healthy Wistar rats (30 males, 30 females) were used in the present study. A rat model of CAG was successfully established using the combined active immunization/ethanol/sodium deoxycholate method. BXD was prepared from a mixture of seven Chinese herbs, and was intragastrically administered to CAG rats at three different doses (6, 12, and 24 g·kg－1·d－1). After 24 weeks, the rats were euthanized, and gastric tissue specimens were collected. Gastric mucosal specimens were stained with hematoxylin and eosin for histological examination to evaluate the degree of inflammation and morphological changes. Immunohistochemical staining was performed to examine the mucosal expression of proliferating cell nuclear antigen. Serum gastrin levels were measured using radioimmunoassay. The expression of Notch signaling-associated genes was examined by quantitative polymerase chain reaction and Western blot assay. RESULTS: BXD at all three doses significantly reversed the adverse effects generated by CAG in rats. Compared with control rats, the CAG rats who were administered BXD had an accelerated growth rate, reduced inflammatory cell infiltration, improved gastric mucosal morphology, augmented thickness of the gastric mucosa, increased number of gastric glands, enhanced mucosal expression of proliferating cell nuclear antigen, and elevated serum gastrin levels. CONCLUSION: BXD has a therapeutic effect in a rat model of CAG by targeting the Notch signaling pathway, thereby blocking the CAG from progressing to early gastric cancer.

Effect of Total Flavone of Haw Leaves on Nuclear Factor Erythroid-2 Related Factor and Other Related Factors in Nonalcoholic Steatohepatitis Rats.

OBJECTIVE: To investigate the effect of total flavone of haw leaves (TFHL) on the expression of nuclear factor erythroid-2 related factor (Nrf2) and other related factors in nonalcoholic steatohepatitis (NASH) rats induced by high-fat diet and then to further discuss the mechanism of TFHL's prevention against NASH. METHODS: High-fat diet was fed to 40 rats to establish the NASH model. Then model rats were intragastrically administrated with 40, 80, 160 mg/(kg•day) TFHL, respectively. The pathological changes of liver tissues in NASH rats were detected by oil red O and hematoxylin-eosin (HE) stainings. The expression of Nrf2 in rat liver was examined through immunohistochemistry. The level of 8-iso-prostaglandin F2α in serum was detected through enzyme linked immunosorbent assay (ELISA). The mRNA and protein levels of Nrf2 and other related factors in liver tissue were measured by real-time reverse transcriptionpolymerase chain reaction and western blot. RESULTS: Lipid deposition, hepatic steatosis, focal necrosis in lobular inflammation and ballooning degeneration were emerged in livers of NASH rats. The 8-iso-prostaglandin F2α in the serum of NASH rats increased significantly compared with the control group (P<0.05). The mRNA of Nrf2, hemeoxyenase1 (HO-1) and the mRNA and protein levels of quinine oxidoreductase (NQO1) in NASH rats liver tissue showed a striking increase, while the mRNA levels of Keap1, r-glutamylcysteine synthethase (rGCS) and glutathione S-transferase (GST) were significantly decreased compared with the control group (P<0.05). After TFHL treatment, 8-iso-prostaglandin F2α level in serum significantly decreased, and Nrf2 mRNA and protein levels in hepatocytes nucleus enhanced compared with the model group (P<0.05 or 0.01). Meanwhile the Keap1 mRNA, the mRNA and protein levels of HO-1, NQO1 antibody, rGCS antibody, GST increased after TFHL treatment (P<0.05 or 0.01). CONCLUSIONS: Nrf2 and other related factors were involved in development of NASH, and they also served as an important part in its occurrence. By regulating expression of Nrf2 and other related factors, TFHL may play a role in antioxidative stress and prevention of NASH.

[Effect of total flavones of hawthorn leafonon expression of COX-2/Nrf2 in liver of rats with nonalcoholic steatohepatitis].

To explore the effect of total flavones from hawthorn leaf on (THFL) on the expression of COX-2/Nrf2 in the liver tissues of rats with nonalcoholic steatohepatitis (NASH), and discuss its anti-NASH mechanism, thirty-two SD rats were randomly divided into the normal group, model group, THFL high dose group and low dose group, 8 in each group. High fat diet was given to the rats for 12 weeks to establish the NASH models, and the high and low dose groups were administered with TFHL at the dosage of 250, 125 mg•kg⁻¹â€¢d⁻¹ respectively. Steatosis and the inflammatory changes of the liver tissues in rats were observed by HE staining; T-AOC level was detected by colorimetry; the level of 8-OHdG and the protein expressions of COX-2, Nrf2 and HO-1 in the liver tissues were determined by immunohistochemistry; and the mRNA expressions of COX-2, Nrf2 and HO-1 in liver tissues were detected by Real time-PCR. Compared with the normal group, the liver steatosis, ballooning degeneration for inflammatory degree and NAFLD activity scores (NAS) were significantly increased in model group, while total antioxidant capacity (T-AOC) was decreased, DNA damage marker 8-OHdG level was increased, and the mRNA and protein expressions of COX-2, Nrf2 and HO-1 were significantly increased. After the administration of high and low dose of TFHL, the inflammation degree of the liver tissues and NAS were significantly decreased, 8-OHdG level and COX-2mRNA and protein expressions were decreased, and the mRNA and protein expressions of Nrf2 and HO-1 were significantly increased when compared with the model group. COX-2/Nrf2 pathway was involved in the development and progression of NASH induced by high fat diet. TFHL could prevent the development of NASH by promoting the expression Nrf2/HO-1, regulating and inhibiting the over expression of COX-2, and further attenuating the cell injury and hepatic inflammation caused by oxidation reaction.

[Experimental study on intervention effect of Grifola frondosa on nonalcoholic steatohepatitis].

To study the preventive effect of Grifola frondosa on nonalcoholic steatohepatitis (NASH). The rat model of NASH was established by feeding high-fat diets for 12 weeks and intervened with 0.5 g · kg(-1) · d(-1) and 1.0 g · kg(-1) · d(-1) of C. frondosa powder suspensions. The degrees of hepatocyte fatty degeneration and inflammation were observed under the optical microscope with routine HE staining. The NAFLD activity scores (NAS) were calculated. Serum ALT, AST and hepatic TG and CHOL were tested by the biochemical method. The hepatic MDA was examined by thiobarbituric acid method. The hepatic SOD was tested by the xanthine oxidase test. The hepatic GSH-PX activity was determined by the dithio-nitrobenzoic acid method. Hepatic TNF-α and IL-6 were detected by the enzyme-linked immunosorbent assay (ELISA). The NASH model group induced by high-fat diets showed higher hepatic NAS, ser- um ALT, AST, CHOL and hepatic TG, CHOL, MDA, TNF-α, IL-6 (P < 0.01 or P < 0.05) and lower serum TG and hepatic SOD, GSH-PX (P < 0.01, P < 0.05) than the normal control group. After being intervened with different doses of G. frondosa, the NASH group revealed significantly lower hepatic NAS, serum ALT and hepatic TG, CHOL, MDA, TNF-α and IL-6 (P < 0.05) and higher hepatic SOD, GSH-PX (P < 0.05) than the model group. G. frondosa may prevent the further development of NASH by improving the disorder of lipid metabolism in rats with NASH induced by high-fat diets, relieving the level of oxidative stress and reducing the generation of inflammatory cytokines.

[Effect of pure total flavonoids from citrus on hepatic SIRT1/PGC-1alpha pathway in mice with NASH].

OBJECTIVE: To observe the effect of pure total flavonoids from Citrus (PTFC) on the hepatic fatty degeneration, inflammation, oxidative stress and SIRT1/PGC-1alpha expressions in mice with non-alcohol steatohepatitis (NASH), and discuss the action mechanism of PTFC on NASH. METHOD: Mice were given high-fat diet for 16 weeks to induce the NASH model. Since the seventh week after the model establishment, the mice were intervened with 100, 50 and 25 mg x kg(-1) x d(-1) PTFC for 10 weeks. The pathologic changes in hepatic tissues were observed with HE staining. The contents of TG, CHOL in hepatic tissue, as well as the levels of AST, ALT in serum were detected by using the biochemical process. The expression of SIRT1, PGC-1alpha and MnSOD mRNA in hepatic tissues were detected with Real-time PCR assay. SIRT1, PGC-1alpha protein and 8-OHdG expressions were determined with the immunohistochemical method. The SOD level in hepatic tissues was tested by the xanthine oxidase method. The MDA content in hepatic tissues was examined by the thiobarbituric acid method. RESULT: The contents of TG, CHOL, NAFLD activity scores and ALT level in serum in hepatic tissues of mice in the model induced by fat-rich diet were obviously higher than that of the normal group (P < 0.010. The SIRT1, PGC-1alpha, MnSOD mRNA and protein expression in hepatic tissues were significantly lower than that of the normal group (P < 0.01). The expression of 8-OHdG and the content of MDA in hepatic tissues were obviously higher than that of the normal group (P < 0.01). After the intervention with different doses of PTFC, the NAFLD activity scores, the content of TG and the level of AST in serum were notably lower than that of the normal group (P < 0.01, P < 0.05); whereas the SIRT1, PGC-1alpha, MnSOD mRNA and protein expression were obviously higher than that of the normal group (P < 0.01, P < 0.05), with the significant decrease in the expression of 8-OHdG and the content of MDA (P < 0.01). CONCLUSION: Oxidative stress/lipid peroxidation enhancement in in NASH mice induced by high-fat diet may be related to the changes in SIRT1/PGC-1alpha signal transduction pathway. PTFC could enhance the anti-oxidant capacity in liver, relieve the damage of reactive oxygen during the fatty acid metabolic process, and prevent NASH from the occurrence and development by regulating the SIRT1/PGC-1alpha signal pathway.

[Effect of total flavones of hawthorn leafon (TFHL) on expression of UCP2 in liver of NASH rats].

OBJECTIVE: To study the expression of uncoupling protein 2 (UCP2) in liver of rats with nonalcoholic steatohepatitis (NASH) induced by fat-rich diet, and the effect of total flavones of hawthorn leafon (TFHL) on UCP2. METHOD: The NASH model of rat was induced by 12 weeks of fat-rich diet. Subsequently the rats were administrated with TFHL in accordance with 250, 125 mg x kg(-1) x d(-1) and the Essentiale N with 195.4 mg x kg(-1) x d(-1). The change of liver pathological. The levels of serum ALT and AST, the content of TG, CHOL, MDA and T-AOC activity of liver and were evaluated. The UCP2mRNA expression in liver was detected with RT-PCR, and the contents of UCP2 were examined with ELISA. RESULT: There are severe steatosis, inflammatory cellular infiltration in the liver of the NASH models. The levels of serum ALT, AST and the contents of TG, CHOL, MDA and UCP2 in the model group were higher than those of in the normal groop. The expression of UCP2mRNA was obviously enhanced and the activity of T-AOC decreased. The expression of UCP2 mRNA of rats was positively correlation with the contents of MDA, TNF-alpha. The inflammation activity in rat liver, the contents of MDA and UCP2, the expression of UCP2 mRNA in the administrated groups were obviously lower than those in the model group, while the activity of T-AOC was higher than that of model. CONCLUSION: TFHL may alleviate liver injury by means of the suppression of Oxidative stress/lipid peroxidation reaction and the overexpression of UCP2 in liver, which could prevent the further development of NASH.

[Influences of FVP1 on the curative and negative effects of CTX].

OBJECTIVE: To observe the influences of FVP1 on both curative and negative effects of CTX. METHOD: The present study included two parts of experiments. In the part 1, 0.2 mL of 1 x 10(7) mL(-1) of S180 cells were inoculated in the subcutaneous layer of the right armpit of mice. All the mice were randomly divided into 3 groups: control group, in which mice were given with normal saline in 10 consecutive days, CTX group, in which mice were injected with 30 mg of CTX in the first and third days and saline in the other 8 days during the 10 consecutive days of treatment, and FVP1 and CTX group, in which the mice were injected with 30 mg x kg(-1) of CTX in the first and third days and FVP1 at 10 mg x kg(-1) in all 10 consecutive days of treatment. After above 10-day treatment , all the mice were killed and the tumor body was taken out and weighed to calculate the inhibiting rates on tumor. In the part 2 of experiments all the mice were divided into 3 groups: Normal control group, in which mice were not treated with any drugs, CTX-induced model group of inhibiting immune system, in which mice were injected with CTX at dose of 10 mg x kg(-1) in first two days and saline in the following 7 days; and small-, meddle-and large-dosage of FVP1 groups, in which mice were injected with CTX at the same dose as above in first two days and FVP1 intraperitoneally at 5, 10 and 20 mg x kg(-1) respectively in the following 7 days. CTX group was regarded as the control model. After the treatment, the peripheral white cells, thymus index, spleen index, the phagocytic power of macrophage of abdominal cavity, lymphocyte trastation rate and the activity of NK cell were detected. RESULT: (DFVP1 plus small dose of CTX obviously enhanced the inhibiting rate of CTX on tumor in the mice inoculated with S180 cells. (2) FVP1 at the different dose obviously antagozized CTX-induced leucopenia, atrophy, reduction of the phagocytic power of macrophage in abdominal cavity and restored the function of lymphocyte translation and the activity of NK cells. CONCLUSION: FPV1 could enhance the curative effect of CTX in depressing tumor and attenuate the negative effect of CTX in inhibiting the function of immune system.