Regulation Of Resolving Stagnation And Phlegm Herbs On The LXRs Signaling Pathway In Rats With Nonalcoholic Fatty Liver Disease

Objective: To observe the effect of Resolving stagnation and phlegm Herbs(hereinafter using abbreviation RSPH) on the content of TC, TG, HDL-C and LDL-C in blood serum and liver of rats with nonalcoholic fatty liver disease(NAFLD); To investigate the influence of RSPH on the expression of LXRαm RNA in liver and its down stream gene SREBP-1c m RNA and CYP7A1 in liver; Further to discusse the therapeutic effect of RSPH in NAFLD and its possible mechanism.Methods: 60 male Sprague-Dawley rats(160-200g) were purchased from the Laboratory Animal Center(Hebei Medical University, China). Rats were housed in standard rat cages and were exposed to a 12-h light: 12-h dark cycle. After 1 week, they were randomly divided into 6 groups that each grou Phad 10 rats: Normal control group, Model control group, High-dose of RSPH group,Middle-dose of RSPH group, Low-dose of RSPH group, Dongbaogantai control group. In the next 8 weeks, rats in normal grou Pwere fed with ordinary forage, and the others were fed with high-fat forage. At the same time, control drug or treatment drug was given to each grou Pand equal amount of Saline was given to normal and model groups which lasted for 4 weeks of stomach-perfusion. Rats in each grou Pwere given stomachperfusion once a day and the drug volume was calculated by 1ml/100 g. 12 hours fasting after the last stomach-perfusion, all rats were anaesthetized and their blood were drawn out from femoral artery, centrifuged, and then isolated the serum in order to measure the content of TC, TG, HDL-C, LDL-C and FFA in blood serum. Simultaneously, the liver was obtained quickly and portion hepatic tissue was removed in right lobe of liver with divided into triplicate, the first one was frozen in-70 ℃for testing the expression of LXRαm RNA, SREBP-1cm RNA, CYP7A1 m RNA. The second one was soaked in 4% paraform, paraffin imbedding, slice, HE stained to provide for the observation of the light microscope.The third one was frozen in-70 for ℃testing the content of TG、TC in liver.Results:1 Content of TC, TG, HDL-C and LDL-C in serum of NAFLD rats in each group Compared with normal group, the contents of TC, TG and LDL-C in serum of model grou P(3.298±0.256, 1.180±0.153, 2.743±0.466, 436.481±52.151) were obviously increased(1.371±0.196, 0.512±0.093, 0.440±0.050,243.335±50.284)(P<0.01), and the content of HDL-C(0.719±0.098) was obviously decreased(1.119±0.186)(P<0.01), which showed that the NAFLD model was made successfully. After the intervention of medicine, every treatment grou Pcould significantly reduce the content of TG, TC, LDL-C and FFA(P<0.01), and increase the content of HDL-C(P<0.01). Among the total, the content of TC(1.484±0.239, 1.690±0.27, 1.741±0.269), TG(0.554±0.098, 0.574±0.093, 0.669±0.105), LDL-C(0.885±0.181, 1.095±0.266, 1.215±0.289) and FFA(242.191±35.130, 259.780±29.330, 277.622±34.284) in three dose groups of RSPH were obviously lower than control grou P(2.131±0.352, 0.827±0.109, 1.593±0.313, 324.051±56.061)(P < 0.05 or P < 0.01). The content of HDL-C(1.480±0.219, 1.296±0.228, 1.305±0.233) in three dose groups of RSPH were obviously higher than control grou P(1.123±0.131)(P<0.05 or P<0.01). The content of TC, TG, LDL-C and FFA in high-dose grou Pwere significantly lower than low-dose group.while the content of HDL-C was higher than middle-dose grou Pand low-dose group(P<0.05). The content of TC, TG, LDL-C and FFA in middle-dose grou Pwere lower than low-dose grou P, the content of HDL-C higher than low-dose grou Pbut it had no statistical significance(P>0.05). 2 Content of TC, TG in liver of NAFLD rats in each group Compared with normal group, the contents of TC, TG in liver of model grou P(15.982±2.366,36.979±4.283) were obviously increased(6.129±0.843, 21.280±3.031)(P<0.01). Compared with model group, every treatment grou Pcould significantly reduce the content of TG, TC in liver(P<0.01). Among the total, the content of TC(7.145±0.819, 8.592±0.948, 9.963±1.044), TG(23.650±3.276, 24.409±3.150, 26.368±3.598)in three dose groups of RSPH were obviously lower than control grou P(10.946±1.871, 29.591±4.376)(P<0.05). RSPH could decrease the content of TC,and the satisfied dose-effect relationshi Pwas investigated among three doses(P<0.05). The content of TG in high-dose grou Pof RSPH was obviously lower than low-dose grou P(P < 0.05), there was no statistical significance between high-dose grou Pthan middle-dose grou P((P>0.05). 3 Morphometric Analysis of Liver Tissue in NAFLD rats In normal group, the structure of hepatic lobules and hepatic sinusoid were clear, hepatic cells arranged orderly and distributed as radial around central veins. The nuclear form was normal, cytoplasm were well-distributed. As for the rats in model group, the hepatic cells arranged disordered and in different sizes. There also appeared obviously fatty degeneration: a lot of different sizes lipid droplets inside the cytoplasm, furthermore, the nucleus deviated from the normal position. Compared with the model group, the pathological changes in each treatment grou Pwere ameliorated in varying degree. The number of lipid droplets and adipose hollow space were decreased obviously, the structure of hepatic lobules and hepatic sinusoid was clearer and the fatty degeneration was improved significantly than model group. 4 Expression of LXRαm RAN in hepatic tissue of NAFLD rats Compared with normal grou P(0.426±0.056), the expression of LXRαm RAN in model grou P(1.337±0.216) were markedly degraded(P<0.01). After the intervention of medicine, each treatment could strengthen the expression of LXRαm RAN(P<0.01). The expression of LXRαm RAN in three dose groups of RSPH(0.537±0.059, 0.605±0.072, 0.691±0.083) were obviously lower than control grou P(0.772±0.052)(P<0.05 or P<0.01). RSPH could down-regulate the expression of LXRαm RAN,and the satisfied dose-effect relationshi Pwas investigated among three doses(P<0.05 or P<0.01). 5 Expression of SREBP-1c in hepatic tissue of NAFLD rats Compared with normal grou P(0.361±0.051), the expression of SREBP-1cm RAN in model grou P(1.026±0.236) were markedly increased(P<0.01). Compared with model group, each treatment could obviously degraded the expression of SREBP-1cm RAN(P<0.01). The expression of SREBP-1cm RAN in three dose groups of RSPH(0.503±0.061, 0.532±0.072,0.581±0.041) were obviously lower than control grou P(0.636±0.033)(P<0.05 or P<0.01). The expression of SREBP- 1cm RAN in high-dose grou Pof RSPH was obviously lower than low-dose grou P(P<0.05), there was no statistical significance between high-dose grou Pthan middle-dose grou P((P>0.05). 6 Expression of CYP7A1 m RAN in hepatic tissue of NAFLD rats Compared with normal grou P(0.782±0.091), the expression of CYP7A1 m RAN in model grou P(0.295±0.044) were markedly degraded(P<0.01). Compared with model group, each treatment could strengthen the expression of CYP7A1 m RAN(P<0.01). The expression of CYP7A1 m RAN in three dose groups of RSPH(0.653±0.071, 0.522±0.063, 0.619±0.081) were obviously higher than control grou P(0.459±0.066)(P<0.01), The expression of CYP7A1 m RAN in high-dose grou Pand low-dose grou Pof RSPH were obviously higher than middle-dose grou P(0.459±0.066)(P<0.01), and there was no statistical significance between high-dose grou Pand low-dose grou P(P>0.05).Conclusions:1 RSPH could significantly reduce the content of TC, TG and LDL-C, and increase the content of HDL-C in serum, showing that it had satisfactory function in regulating blood fat and improving disorder of blood fat.2 RSPH could obviously improve the lesion degree of liver tissue of NAFLD rats, it manifested like this: after the drug was given, the fatty degeneration improved significantly, the number of lipid droplets and adipose hollow space decreased obviously,. 3 RSPH could inhibit the expression of LXRαm RAN in hepatic tissue, it could reduce the liver fat deposition in hepatic tissue, ease and prevent NAFLD pathological changes. 4 RSPH could inhibit the expression of SREBP-1c in hepatic tissue, reduce fat synthesis and the liver fat deposition in hepatic tissue, which could avoid the form of fatty liver. 5 RSPH could strength the expression of CYP7A1 m RAN in hepatic tissue, promote cholesterol transform into bile acid, and enhance metabolism and excretion of cholesterol to achieve the purpose of curing the HLP.