参苓白术散对NAFLD大鼠肝脏超微结构及mTOR、STAT3蛋白磷酸化的影响

目的:观察参苓白术散对非酒精性脂肪性肝病(NAFLD)大鼠肝组织mTOR、STAT3蛋白磷酸化的影响。方法:SPF级SD大鼠40只随机分为正常对照组、模型组及参苓白术散低、高剂量组,每组10只。采用高脂饲料喂养复制大鼠NAFLD模型组,8 w后处死大鼠,取血、肝组织。全自动生化分析检测血清TC、TG、ALT、AST,ELISA法检测血清IL-6、TNF-α水平,HE染色和油红O染色观察肝组织病理学改变,透射电子显微镜观察肝组织超微结构变化,Western blot法检测大鼠肝组织mTOR、STAT3蛋白磷酸化水平。结果:8 w高脂饮食成功复制了NAFLD大鼠模型,模型组大鼠肝组织存在严重的脂质蓄积和肝脏脂肪变性,血清TNF-α、IL-6含量及肝组织p-mTOR/mTOR、p-STAT3/STAT3水平较正常对照组显著升高(P<0.01)。与模型组比较,各给药组血脂及病理组织学有不同程度改善,血清TC、TG、ALT、AST、TNF-α、IL-6水平及肝组织mTOR、STAT3蛋白磷酸化水平显著降低(P<0.05或P<0.01)。参苓白术散高剂量作用优于低剂量。结论:mTOR、STAT3蛋白可能是参苓白术散防治NAFLD的有效作用靶点。参苓白术散可能通过抑制肝组织mTOR、STAT3蛋白磷酸化,减轻肝脏脂质蓄积及炎症反应,发挥防治NAFLD的作用。

Effect of Soothing Gan(Liver) and Invigorating Pi(Spleen) Recipes on TLR4-p38 MAPK Pathway in Kupffer Cells of Non-alcoholic Steatohepatitis Rats

Objective: To investigate the mechanism of inflammatory-mediated toll-like receptor 4(TLR4)-p38 mitogen-activated protein kinase(p38 MAPK) pathway in Kupffer cells(KCs) of non-alcoholic steatohepatitis(NASH) rats and the intervention effect of soothing Gan(Liver) and invigorating Pi(Spleen) recipes on this pathway. Methods: After 1 week of acclimatization, 120 Sprague-Dawley male rats were randomly divided into 8 groups using a random number table(n=15 per group): normal group, model group, low-dose Chaihu Shugan Powder(柴胡疏肝散, CHSG) group(3.2 g/kg), high-dose CHSG group(9.6 g/kg), low-dose Shenling Baizhu Powder(参苓白术散, SLBZ) group(10 g/kg), high-dose SLBZ(30 g/kg) group, and low-and highdose integrated recipe(L-IR, H-IR) groups. All rats in the model and treatment groups were fed with a high-fat diet(HFD). The treatments were administrated by gastrogavage once daily and lasted for 26 weeks. The liver tissues were detected with hematoxylin-eosin(HE) and oil red O staining. Levels of liver lipids, serum lipids and transaminases were measured. KCs were isolated from the livers of rats to evaluate the mRNA expressions of TLR4 and p38 MAPK by real-time fluorescence quantitative polymerase chain reaction, and proteins expressions of TLR4, p-p38 MAPK and p38 MAPK by Western blot. Levels of inflammatory cytokines including tumor necrosis factor α(TNF-α), interleukin(IL)-1 and IL-6 in KCs were measured by enzyme-linked immunosorbent assay. Results: After 26 weeks of HFD feeding, HE and oil red O staining showed that the NASH model rats successfully reproduced typical pathogenesis and histopathological features. Compared with the normal group, the model group exhibited significant increases in body weight, liver weight, liver index, serum levels of total cholesterol(TC), triglyceride(TG), low-density lipoprotein cholesterol, and aspartate aminotransferase as well as TC and TG levels in liver tissues, and significant decrease in serum level of high-density lipoprotein cholesterol(P<0.05 or P<0.01), while those indices were significantly ameliorated in the H-IR group(P<0.05 or P<0.01). Higher levels of TNF-α, IL-1 and IL-6 in KCs were observed in the model group compared with the normal group(P<0.01). Significant decreases in TNF-α, IL-1 and IL-6 were observed in the H-SLBZ, H-IR and L-IR groups compared with the model group(P<0.05 or P<0.01). The m RNA expressions of TLR4 and p38 MAPK and protein expressions of TLR4, p38 MAPK and p-p38 MAPK in KCs in the model group were significantly higher than the normal group(P<0.01), while those expression levels in the L-IR and H-IR groups were significantly lower than the model group(P<0.05 or P<0.01). Conclusions: Inflammation in KCs might play an important role in the pathogenesis of NASH in rats. The data demonstrated the importance of TLR4-p38 MAPK signaling pathway in KCs for the anti-inflammatory effect of soothing Gan and invigorating Pi recipes.

Ginsenosides from Stems and Leaves of Ginseng Prevent Ethanol-Induced Lipid Accumulation in Human L02 Hepatocytes

Objective： To investigate the effect of ginsenosides from stems and leaves of ginseng on ethanolinduced lipid deposition in human LO2 hepatocytes. Methods： LO2 cells were exposed to ethanol for 36 h and treated with or without ginsenosides. The viability of LO2 cells was evaluated by methylthiazolyldiphenyl- tetrazolium bromide assay and the triglyceride （TG） content was detected. Lipid droplets were determined by oil red O staining. Intracellular reactive oxygen species （ROS） production and the mitochondrial membrane potential were tested by flow cytometry. The ATP level was measured by reverse phase high performance liquid chromatography. The expression of cytochrome p450 2E1 （CYP2E1） and peroxisome proliferator- activated receptor （PPAR α） was detected by reverse transcriptase-polymerase chain reaction and Western blotting, respectively. Results： Ethanol exposure resulted in the increase of TG level, lipid accumulation and ROS generation, and the decrease of mitochondrial membrane potential and ATP production in the cells. However, ginsenosides significantly reduced TG content （9.69 ± 0.22 μg/mg protein vs. 4.93 ± 0.49 μg/mg protein, P〈0.01）, and ROS formation （7254.8± 385.7 vs. 5825.2± 375.9, P〈0.01）. Meanwhile, improvements in mitochondrial membrane potential （10655.33± 331.34 vs. 11129.52 ± 262.35, P〈0.05） and ATP level （1.20±0.18 nmol/mg protein vs. 2.53±0.25 nmol/mg protein, P〈0.01） were observed by treatment with ginsenosides. Furthermore, ginsenosides could down-regulate CYP2E1 expression （P〈0.01） and upregulate PPAR α expression （P〈0.01） in ethanol-treated cells. Conclusions： Ginsenosides could prevent ethanol-induced hepatocyte steatosis in vitro related to the inhibition of oxidative stress and the improvement of mitochondrial function. In addition, the modulation of CYP2E1 and PPAR α expression may also play an important role in the protective effect of ginsenosides against lipid accumulation.