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    [Effects of soothing liver and invigorating spleen recipes on NF-kappaB signal pathway related genes and proteins in primary hepatocytes of rats with NASH]. Yang Qin-He,Xu Yong-Jian,Feng Gao-Fei,Yang Xue-Song,Han Li,Zhang Yu-Pei,Wang Hong Zhong yao cai = Zhongyaocai = Journal of Chinese medicinal materials 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.
    Effects of extracts from soothing-liver and invigorating-spleen formulas on the injury induced by oxidative stress in the hepatocytes of rats with non-alcoholic fatty liver disease induced by high-fat diet. Tu Guifang,Gong Xiangwen,Yang Qinhe,Li Yuanyuan,Wang Guanlong,Liang Yinji,Zhang Yupei,Yan Haizhen,Lin Chunmei,Zhang Jinwen Journal of traditional Chinese medicine = Chung i tsa chih ying wen pan OBJECTIVE:To investigate effects of the extracts from soothing-liver and invigorating-spleen formulas on the injury due to oxidative stress, mediated by the Nuclear factor-like 2 (Nrf2)-Antioxidant response element (ARE) pathway, in the hepatocytes of rats with non-alcoholic fatty liver disease (NAFLD) induced by high-fat diet. METHODS:Soothing-liver and invigorating-spleen formula mixtures were prepared for five groups: normal, model, soothing-liver formula group (SLG), invigorating-spleen formula group (ISG), integrated formula group (IG). The rat model of NAFLD was induced by feeding rats a high-fat diet (HFD). After 16 weeks, the hepatic tissue was examined following Haematoxylin-Eosin (H&E) staining and with Transmission electron microscopy (TEM). Levels of hepatic lipids, serum lipids and hepatic functions were measured using a biochemical analyser. Hepatocytes were isolated from the livers of rats and were identified by cellar immunohistochemistry, cellular immunofluorescence and flow cytometry. The expression levels of Nrf2, Kelch-like epichlorohydrin-associated protein 1 (Keap-1), haeme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) mRNAs were assessed by real-time fluorescence quantitative PCR. Nrf2, Keap-1, HO-1 and NQO1 proteins were measured by Western blotting. Finally, the levels of oxidative stress factors Superoxide Dismutase (SOD), malonaldehyde (MDA) and Glutathione peroxidase (GSH-Px) in hepatocytes were measured by WST-1, TBA and colorimetry. RESULTS:The H & E and TEM results showed that the NAFLD model rats successfully reproduced typical pathogenetic and histopathological features. The liver function and levels of hepatic lipids and serum lipids from the model rats were dramatically increased. Compared with the model group, the levels of hepatic lipids, serum lipids and hepatic function in the treatment groups were ameliorated to different degrees. The yields of purified hepatocytes in each rat were 4-5 ¡Á 108. The viability of the isolated hepatocytes was higher than 95%, with a purity over 93.2%. Cellular immunohistochemistry analysis showed that the hepatocytes were brown, while in the cellular immunofluorescence analysis, the hepatocytes showed green fluorescence. The expression levels of Nrf2, Keap-1, HO-1 and NQO1 mRNA and protein in the hepatocytes were significantly higher in the model group than in the normal group (P < 0.05, P < 0.01). Compared with the model group, the expression of Nrf2, Keap-1, HO-1 and NQO-1 mRNAs and proteins in all treatment groups increased, especially in the IG (P < 0.01). CONCLUSION:The extracts from soothing-liver and invigorating-spleen formulas may protect the liver against the injury induced by oxidative stress in hepatocytes by influencing the Nrf2-ARE pathway, which may be the mechanism having the potential for prevention and treatment of NAFLD.
    [Effects of soothing liver and invigorating spleen recipes on LPS-induced hepatocytes injury of rats and TLR4/p38MAPK signal pathway]. Gong Xiang-Wen,Yang Qin-He,Yan Hai-Zhen,Zhang Yu-Pei,Liang Yin-Ji,Liu Yi-Zhen,Zhang Jin-Wen ,Lin Chun-Mei,Li Yuan-Yuan Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica OBJECTIVE:To explore the effects of soothing liver and invigorating spleen recipes on lipopolysaccharide(LPS) induced hepatocyte inflammation of rats and TLR4/p38MAPK signal pathway. METHOD:The hepatocytes of SD rats were cultured and identified in vitro. The medicated serum of soothing liver and invigorating spleen recipes was prepared. The hepatocytes were treated with soothing liver and invigorating spleen recipes. Then Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression in cultural supernatants were assayed by ELISA. The expressions of Toll-Like 4 (TLR4), p38 mitogen activated protein kinases (p38MAPK) and p-p38 mitogen-activated protein kinase (p-p38MAPK) were detected by Western blot. RESULT:The rat medicated serum of soothing liver and invigorating spleen recipes was extracted for 2-3 mL. The purified rat hepatocytes were 1.5 x 10(8)-2.0 x 10(8). The cell viability was above 95% detected by Typan blue staining. The hepatocytes were identified by immumofluorescence assay. The detection of hepatocyte cultural supernatants: compared with that of the control group, IL-6 and TNF-α expression were increased in the LPS group (P < 0.01). While compared with that of the LPS group, the expressions of IL-6 and TNF-α were decreased after soothing liver and invigorating spleen recipes intervention (P < 0.01). The detection of hepatocyte proteins: compared with that of the control group, the protein expressions of p38MAPK, p-p38MAPK and TLR4 were all increased significantly in the LPS group (P < 0.01). Compared with that of the LPS group, the protein expressions of p38MAPK was decreased significantly in SB239063 group and it was also decreased in the soothing liver and invigorating spleen recipes group, but with no significant difference. Compared with that of the LPS group, p38MAPK expression was reduced significantly in the soothing liver and invigorating spleen recipes group and the SB239063 (p38MAPK pathway inhibitor) group (P < 0.01). TLR4 protein expression was decreased markedly in the soothing liver and invigorating spleen recipes group (P < 0.01) but had no difference between the SB239063 group and the LPS group. CONCLUSION:The soothing liver and invigorating spleen recipes may regulate hepatocyte inflammatory injury of rats through TLR4/p38MAPK signaling pathway.