| Background:Non-alcoholic fatty liver disease(NAFLD) is a lipid metabolism disorder character byliver cell fatty degeneration and non-alcoholic steatohepatitis. In recent year, the NAFLDincidence rapidly rise, but we lack of effective measures to treat this disease. Thetraditional Chinese medicine have some advantages and effectives to cure this disease. Itwould be a feasible step to use molecular biology technology to find out the mechanism ofNAFLD which is therapy by TCM. As a previous research achievement by professorchangquan Ling of changhai hospital, which he exploit a medicine, named ganzaoning,have some effectives to prevent and cure NAFLD. Ganzaoning is formed by Chineseherbal medicines, which conclude Chinese yam. Our research is using the main componentof Chinese yam. It’s named Diosgenin. We want to research it anti NAFLD mechanism.Objective:From theAMPK cell signaling pathway and it’s relative protein and genes to discussthe diosgenin anti NAFLD through the vivo and vitro experiments.Methods:In vitro experiments（1） We use HepG2cell for our study, which culture by10%FBS low glucose(glucosecontents:1.0g/L) DMEM solution. We digest and remove it to the new6wells plate,hunger for12h with no FBS low glucose. Then we stimulate HepG2cells24h usingby high glucose(glucose contents:4.5g/L) and different concentration diosgenin(0、5、10、25、50、100μΜ). After that, we collect the protein of HepG2cells for Westernblotto detect AMPK andACC protein expression.（2） We use HepG2cell for our study, which culture by10%FBS low glucose(glucosecontents:1.0g/L) DMEM solution. We digest and remove it to the new6wells plate,hunger for12h with no FBS low glucose. Then we stimulate HepG2cells24h usingby high glucose(glucose contents:4.5g/L) and different concentration diosgenin(0、5、10、25、50、100μΜ).After that, we use the TG kit to detect HepG2cell TGconcentration.（3） We use HepG2cell for our study, which culture by10%FBS low glucose(glucosecontents:1.0g/L) DMEM solution. We digest and remove it to the new6wells plate,hunger for12h with no FBS low glucose. Then we stimulate HepG2cells24h using by high glucose(glucose contents:4.5g/L) which contains1μM Compound C and25μM Diosgenin. After that, we use the TG kit to detect HepG2cell TG concentration.（4） We use HepG2cell for our study, which culture by10%FBS low glucose(glucosecontents:1.0g/L) DMEM solution. We digest and remove it to the new6wells plate,hunger for12h with no FBS low glucose. Then we stimulate HepG2cells24h usingby low glucose(glucose contents:1g/L) which contains1μM T090137,1μMCompound C and25μM Diosgenin. After that, we use the TG kit to detect HepG2cellTG concentration.In vivo experiments（1） Firstly,40adult SD rats are divided in to5groups, which contains blank group,model group, low concentrate diosgenin, high concentrate diosgenin, fenofibrate group.Each groups have8rats.The blank group use ordinary diet to feed, the other groupsuse high fat diet feeding for16weeks. From them, the low concentrate diosgeningroup use high fat diet which1kg diet contain5g diosgenin, the high concentratediosgenin group use high fat diet which contain1kg diet contain10g diosgenin.Fenofibrate group use high fat diet which1kg diet contain200mg fenofibrate.All ofgroups raise for16weeks. Each rat eat25g/d.（2） After16weeks.we scale rats weight, absorb eyeball blood. Take out the liver ofrats for study.（3） Use oil red stain to detect the rats liver cell changes. Use HE stain to detect therats liver cell changes.（4） Use RT-PCR to detect the rats livers genes which about SREBPs and PPARs genesexpressions.（5） Use Westernblot to detect the rats livers protein which about AMPK,ACC andLXRα expressions（6） Use the automatic biochemical analyzer to detect rats serum which conclude AST,ALT, TG,TC,HDL and LDL.Result:In vitro experiments:（1） After use high glucose DMEM to stimulate the HepG2cells is significant higher(P<0.01) than measured in some concentrate diosgenins at the level of TG, the differencehave statistical significance (P<0.01). （2） Western Blot find that Diosgenin can effectively increaseAMPK phosphorylationand ACC phosphorylation.（3） using the Compoud C and T090317to measure TG levels by TG kits, we canfind that Diosgenin would through AMPK and LXRα to adjust HepG2cells TG levels.In vivo research（1） In the Oil Red O staining and HE staining, we find that fatty degeneration ofmodel liver cells is proved successful. Diosgenin groups compare with model group, theeffective inhibition of hepatocyte TG deposition and reduce hepatic steatosis are obviously.（2） Compared with the control group, the model group serum TG levels aresignificantly increased (P<0.01); Fenofibrate serum TG levels are significantly lower thanthe model group, the difference have statistical significance (P<0.01), the high diosgeninand low-dose group serum TG levels are significantly lower than that in the model group,the difference have statistical significance (P<0.01).（3） Compared with the control group, the model group is significant elevate liverprotein TG ratio (P<0.01); Fenofibrate group TG protein is lower than the model group, thedifference have statistical significance (P<0.01); low doses diosgenin group lower thanfenofibrate group, which have statistical significance (P<0.01).（4） Compared with the control group, the model group SREBP1C expression issignificant increased (P<0.01). Each of diosgenin groups SREBP1C expression lower thanin the model group, the difference have statistical significance (P<0.01);（5） Compared with the control group, the model group PPARα expression is significantdecreased, the difference have statistical significance (P<0.05), PPARα expression levelare of diosgenin groups higher than in the model group, the difference have statisticalsignificance (P <0.01). The PPARα of fenofibrate group expression is significant higherthan the model group, the difference have statistical significance (P<0.01).（5） Compared with the control group, the model group AMPK phosphorylation levelis suppressed. Low diosgenin group AMPK phosphorylation level is higher than the modelgroup. Compared with the control group, the model group LXRα protein level is increased.The each diosgenin groups LXRα protein level are lower than model group.（6） Compared with the control group, the model group ALT expression is significantincreased, the difference have statistical significance (P<0.01). Fenofibrate ALT level issignificant higher than the model group, the difference have statistical significance(P<0.01), low diosgenin group ALT is significant lower than in the model group, the difference is significant (P<0.01), each of diosgenin groups are significantly lower than inthe fenofibrate group, the difference are statistical significant (P<0.01).（7） Compared with the control group, model group body weight, liver weight areincreased, the difference have statistical significance (P<0.01). Each of diosgenin groupsbody weight and liver weight is lower than the model group, the difference have statisticalsignificance (P<0.01).Conclusion:（1） Diosgenin can inhibit the NAFLD in rats which feed by high fat diet.（2） Diosgenin show it anti NAFLD effects would through activeAMPKphosphorylation andACC phosphorylation.meanwhile, Diosgenin would adjust SREBP1Cand PPARα to inhibit TG accumulations.（3） Diosgenin show no obvious harm to liver functions. |
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