| ChapterⅠ The effect and mechanism of astragaloside IV in the treatment of NAFLD mice via 5-LO/LTB4 pathwayObjective:To evaluate the therapeutic effect of Astragaloside IV(ASIV)on non-alcoholic fatty liver disease(NAFLD)in mice,and to explore whether this effect is related to5-lipoxygenase/Leukotriene pathway.Methods:1.40 male Kunming mice weighing about 20-22 g were randomly divided into control group(Control),high-fat diet group(High-Fat diet,HFD),HFD+ASIV low-dose group(20 mg/kg),HFD+ASIV medium-dose group Group(40 mg/kg),HFD+ASIV high-dose group(80 mg/kg),and received a different diet within 16 weeks,each group has 8 animals.The mice were fed HFD for 16 week,and the control animals were fed a standard rodent diet.Three weeks later,the HFD-fed mice were given different doses of ASIV by gavage.The control mice received the same volume of 0.5 % sodium carboxymethyl cellulose(CMC),and their body weight was measured every week.After 16 weeks,all mice were given different doses of ASIV.The animals were euthanized and the wet weight of the liver was recorded.2.Liver morphology: The collected liver tissue fragments were sliced and stained with hematoxylin and eosin staining(HE)to observe the pathological damage of liver tissue in mice.3.Detection of triglyceride(TG),alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels in serum: use TG,ALT and AST kits to test the content of TG,ALT and AST in serum of mice in control group,HFD group and ASIV low,medium and high group.4.ELISA detection of leukotriene B4(Leukotriene,LTB4),tumor necrosis factor(TNF-α)and interleukin 6(Interleukin,IL)in the serum of mice in the control group,HFD group and ASIV low,medium and high groups-6)content.5.Gene level analysis: q-PCR detection of 5-LO and Leukotriene A4 hydrolase(LTA4H)mRNA expression changes in the liver tissues of mice in the control group,HFD group and ASIV low,medium and high groups.6.Protein level analysis: Western blot detection of 5-LO and LTA4 H protein expression changes in liver tissues of mice in the control group,HFD group,and ASIV low,medium,and high groups.Results:1.After 6 weeks of intervention,compared with the control group,the high-fat diet in the HFD group increased the weight of the mice(P<0.001).After 12 weeks of intervention,ASIV treatment significantly reduced weight gain compared with the HFD group.Among them,mice in the ASIV high-dose group lost the most weight(P<0.001).After 16 weeks of ASIV treatment,compared with the HFD group,the liver weight of mice in the ASIV dose groups decreased to varying degrees of which the ASIV high-dose group decreased the most(P<0.001).2.Compared with the control group,the serum TG,ALT and AST levels of the HFD group were significantly increased(P<0.001),and each dose group of ASIV could reduce the serum ALT、AST and TG in the mice to varying degrees,among which high-dose ASIV reduces the highest level.3.The HE slices showed that the liver cells of the control group mouse liver cords were arranged radially along the central vein,while the HFD group mouse liver slices showed a large amount of lipid accumulation in the liver with obvious fatty vacuoles.The slices of mice in ASIV low,medium and high doses group showed different degrees of reduction in fat vacuoles.4.Compared with the control group,the serum levels of TNF-α and IL-6 in the HFD group increased significantly(P<0.001).Compared with the HFD group,the ASIV low,medium,and high dose groups decreased the contents of TNF-α and IL-6 in the mice to varying degrees,and the high-dose group was the best(P<0.001).5.Compared with the control group,the expression of 5-LO(P<0.001)and LTA4H(P<0.01)mRNA in the liver of the HFD group increased significantly,while the expression of 5-LO and LTA4 H mRNA in mouse liver decreased to varying degrees in the ASIV low,medium,and high dose groups.6.Compared with the control group,the expression of 5-LO and LTA4 H protein in the liver of the HFD group was significantly increased,while the low,medium,and high-dose ASIV groups reduced the 5-LO and LTA4 H protein expression.7.Compared with the control group,the expression of LTB4 in the serum of mice in the HFD group was significantly increased(P<0.001),while the low,medium,and high-dose ASIV groups reduced the expression of LTB4 in the serum of mice to varying degrees.Conclusion:1.ASIV in the low,medium and high dose groups can significantly reduce the body weight and wet liver weight of NAFLD model mice,and improve liver pathological damage in NAFLD model mice,showing that ASIV has the effect of preventing and treating NAFLD mice.2.The protective effect of ASIV on NAFLD model mice may be related to the reduction of serum inflammatory factors TNF-α and IL-6 levels in NAFLD model mice.3.The protective effect of ASIV on NAFLD model mice may be related to the reduction of LTB4 content in mouse serum.4.ASIV may protect NAFLD model mice by down-regulating 5-LO and LTA4 H gene and protein expression in liver tissues of NAFLD model mice.ChapterⅡProtective effect and mechanism of astragaloside IV on PA-induced LO2 cellsObjective:To evaluate the protective effect of ASIV on LO2 cells induced by palmitic acid(PA),to explore whether it involves lipid accumulation,oxidative stress and apoptosis-related effects.Methods:1.The MTT method tests the effect of ASIV with a concentration range of 0-200μg/ml on cell viability.Determine the appropriate concentration of ASIV.2.Oil red O staining and determination of intracellular TG and total cholesterol(total cholesterol,TC)content to assess lipid accumulation in liver cells.3.Changes in oxidative stress levels: ROS,MDA and GSH-Px kits detect the levels of ROS,MDA and GSH-Px in LO2 cells.4.Liver cell apoptosis: Annexin V-FITC/PI double staining of LO2 cells,flow cytometry to observe the liver cell apoptosis.5.Protein level analysis: Western blot detection of Bax and Bcl-2 protein expression in liver cells.Result:1.The effect of ASIV in the concentration range of 0-200 μg/ml on cell viability was tested.The results showed that ASIV treatment did not show any cytotoxicity in liver cells up to 100 μg/m L,but at a concentration of 200 μg/m L ASIV,cell viability decreased significantly(P<0.01).Therefore,we chose 25 μg/ml,50μg/m L and 100 μg/m L ASIV to treat PA-induced LO2 cells.2.Compared with the control group,lipid droplets was significantly increased in PA-induced LO2 cells.The addition of ASIV to PA-induced cells can significantly reduce lipid accumulation.Consistently,compared with the control group,the content of TG and TC in the cells was significantly increased by PA treatment(P<0.001).However,the levels of TCand TG in each dose group of ASIV were lower than those of the PA group to varying degrees.3.Compared with the control group,PA exposure significantly increased the content of ROS(P<0.001)and MDA(P<0.01),while the activity of GSH-Px(P<0.001)was significantly reduced.However,incubation with ASIV significantly increased eliminate these trends.4.Compared with the control group,PA significantly increased the apoptosis rate of hepatocytes(P<0.001),while incubation with ASIV significantly reduced the apoptosis rate(P<0.01).5.Compared with the control group,incubation with PA can up-regulate the expression level of Bax protein(P<0.01),while down-regulate the expression level of Bcl-2 protein(P<0.01).Incubation with ASIV can alleviate this trend(P<0.01).Conclusion:1.ASIV treatment did not show any cytotoxicity in liver cells up to 100 μg/m L,but at a concentration of 200 μg/m L ASIV,the viability of LO2 cells was significantly reduced.Therefore,we chose 25 μg/ml,50 μg/m L and 100 μg/m L ASIV to treat LO2 cells.2.The protective effect of ASIV on PA-treated LO2 cells may be related to its inhibition of LO2 cells secreting TG and TC.3.The protective effect of ASIV on PA-treated LO2 cells may be related to the reduction of ROS,MDA and the increase of GSH-px production in LO2 cells.4.The protective effect of ASIV on PA-treated LO2 cells may be related to the up-regulation of Bcl-2 and down-regulation of the expression of Bax protein in LO2 cells.In summary,the protective effect of ASIV on PA-induced LO2 cells may involve multiple pathways such as lipid accumulation,oxidative stress,and apoptosis. |
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