Effects of Chaihu-Shugan-San and Shen-Ling-Bai-Zhu-San on p38 MAPK Pathway in Kupffer Cells of Nonalcoholic Steatohepatitis.

This study aimed to investigate the effects of Chaihu-Shugan-San (CSS), Shen-Ling-Bai-Zhu-San (SLBZS), and integrated recipe of the above two recipes on inflammatory markers and proteins involved in p38 MAPK pathway in Kupffer cells of NASH rats induced by high fat diet (HFD). Rats were administered at low or high dose of CSS, SLBZS, and integrated recipe except normal group and model group for 16 weeks. The levels of hepatic lipid, TNF- α , IL-1, and IL-6 in liver tissues were measured. Kupffer cells were isolated from livers to evaluate expressions of TLR4, p-p38 MAPK, and p38 MAPK by Western blotting. The results showed that the NASH model rats successfully reproduced typical pathogenetic and histopathological features. Levels of hepatic lipid and liver tissues inflammatory factors in high-dose SLBZS group and integrated recipe group were all lower than that of model group decreased observably. Expressions of TLR4, p-p38 MAPK, and p38 MAPK in Kupffer cells were decreased in all treatment groups, but there was no significant difference between treatment groups. The high-dose SLBZS group had the lowest expression levels of TLR4, and the most visible downtrend in the expression levels of p-p38 MAPK and p38 MAPK was found in the high-dose integrated recipe group. The ratio of p-p38 MAPK to total p38 MAPK protein was obviously increased in all treatment groups. Therefore, our study showed that the activation of p38 MAPK pathway in Kupffer cells might be related to the release of inflammatory factors such as TNF- α , IL-1, and IL-6 in NASH rats. High dose of SLBZS and integrated recipe might work as a significant anti-inflammatory effect in Kupffer cells of NASH rats induced by HFD through suppression of p38 MAPK pathway. It indicated that p38 MAPK pathway may be the possible effective target for the recipes.

[Effects of soothing liver and invigorating spleen recipes on the mRNA and protein expression of TLR4 in hepatic tissues of rats with NASH].

OBJECTIVE: To research the effects of soothing liver and invigorating spleen recipes on expression of TLR4 mRNA and protein expression in hepatic tissue of rats with non-alcoholic steatohepatitis and its mechanism. METHODS: 72 male SD rats were randomly divided into 8 groups: normal group, model group, high-dose soothing liver group (receiving gavage of Chaihu Shugan Powder 9. 6 g/kg), low-dose soothing liver group (receiving gavage of Chaihu Shugan Powder 3.2 g/kg), high-dose invigorating spleen group (receiving gavage of Shen Ling Baizhu Powder 30 g/kg), low-dose invigorating spleen group (receiving gavage of Shen Ling Baizhu Powder 10 g/kg), high-dose integrated Group (receiving gavage of Chaihu Shugan Powder and Shen Ling Baizhu Powder combination recipes 39.6 g/kg), low-dose integrated Group (receiving gavage of Chaihu Shugan Powder and Shen Ling Baizhu Powder combination recipes 13.2 g/kg), 9 rats were in each group. Used high fat diet (10 mL/kg) to establish experiment model of NASH rat. At the end of the sixteenth weeks, the levels of serum lipids, liver lipids and serum aminotransferase were measured by automatic biochemical analyzer; Liver pathology was analyzed by HE and Oil red O staining; TLR4 mRNA was assayed by real-time fluorescent quantitative polymerase chain reaction (RT Q-PCR); TLR4 protein was detected by Western blot. RESULTS: Compared with normal group, the levels of TC, LDL-C in the serum, TC,TG as well as the expression of TLR4 mRNA and protein in the hepatic tissue were dramatically increased in model group (P < 0.01). Compared with the model group, the levels of serum lipids, liver lipids, the expression of TLR4 mRNA and protein in the hepatic tissue were decreased in each treatment group (P < 0.05 or P < 0.01). CONCLUSION: Soothing liver and invigorating spleen recipes can inhibit hepatic TLR4 expression, that may be one of their therapeutic mechanisms. There is much difference between high-does and low-does treatment groups in various testing items, which shows that there is does-effect relationship in intervention NASH.

[Effects of soothing liver and invigorating spleen recipes on NF-kappaB signal pathway related genes and proteins in primary hepatocytes of rats with NASH].

OBJECTIVE: To observe the effects of soothing liver and invigorating spleen recipes on NF-kappaB signal pathway related genes and proteins in primary hepatocytes of rats with NASH. METHODS: SD male rats were randomly divided into 8 groups: normal, model, high/low-dose soothing liver group, high/low-dose invigorating spleen group, high/low-dose integrated group. 15 rats in each group. The NASH model rats were induced by feeding high-fat diet (HFD). The treatment lasted for 16 weeks. Then TC, TG in the liver tissue and serum were determined with automatic biochemical analyzer. HE staining and oil red O staining were operated to observe the pathological changes. Another 6 rats of each group were taken respectively and collagenase (Type IV) was perfused to digest liver tissue with the circulation in vitro to separate hepatocytes. The expression levels of IKK(beta), NF-kappaB mRNA, proteins and phosphorylated IKK(beta) protein in hepatocytes of rats from each group were detected by Real-time Q-PCR and Western Blotting, respectively. RESULTS: Compared with normal group, liver histopathology was changed and levels of TC and TG were elevated in model group indicating hepatocytes had lipid accumulation and lipid metabolic disturbance obviously; The levels of serum TC, and hepatic homogenate TC, TG as well as the expression of IKK(beta) NF-kappa-B mRNA, proteins and phosphorylated IKK(beta) protein in hepatocytes were dramatically increased in model group (P < 0.01). Compared with the model group, the levels of IKK(beta), NF-kappaB mRNA expression were decreased most significanly in the invigorating spleen (with high dose) group and the integrated group (with high dose) (P < 0.01 or P < 0.05). The expression levels of the IKK(beta), NF-kappaB proteins and the phosphorylated IKK(beta) protein in hepatocytes were decreased significaniy in the treatment groups (P < 0.01 or P < 0.05), especially for the invigorating spleen (with high dose) group and the integrated (with high dose). CONCLUSION: Soothing liver and invigorating spleen recipes have effect on NASH rats induced by HFD and its mechanism may be related to the suppression of IKK(beta)/NF-kappaB signal pathway related genes and proteins. And the effect probably has a dose response relationship.

[Effect of soothing liver and invigorating spleen recipes on SREBP-1c mRNA and protein expression in hepatic tissues of rats with non-alcoholic fatty liver disease].

OBJECTIVE: To investigate the effects of soothing liver and invigorating spleen recipes on expression levels of Sterol Regulatory Element-binding Protein-1c (SREBP-1c) mRNA and SREBP-1c protein in hepatic tissue of rats with non-alcoholic fatty liver disease (NAFLD). METHODS: Fifty-five SD rats were randomized into 5 groups: normal control group, model group, soothing liver group, invigorating spleen group and combination group. Except the normal group, the rats in model group and other treatment groups were fed with high-fat emulsion to induce NAFLD. The treatment groups were administered with respective traditional chinese medicine, the normal group and model group received correspondence volume distilled water. After treatment for 8 weeks, the rats were executed to obtain the liver for observing hepatic pathological changes. Expression levels of SREBP-1c mRNA and SREBP-1c protein in hepatic tissues were assayed using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry respectively. RESULTS: Compared with the normal group, the expression level of SREBP-1c mRNA and SREBP-1c protein in rat hepatic tissues of model group was significantly increased (P<0.01). Compared with the model group,the expression levels of SREBP-1c mRNA and SREBP-lc protein in the treated groups was decreased (P<0.01, P<0.05). Specially, expression levels of SREBP-1c mRNA were the lowest in soothing liver group and invigorating spleen group and hepatic fatty changes were the slightest. CONCLUSION: Soothing liver and invigorating spleen recipes can inhibit hepatic SREBP-1c expression. That may be one of their therapeutic mechanisms.