Study On The Mechanism Of Kidney Yang Deficiency And Its Effects On Blood Lipid In Rats

Objects: This research based on the kidney’s main function is gasification,using the intramuscular injection of hydrocortisone established deficiency ofkidney-yang animal models as a platform, to observe the changes of bloodlipid of the rats with kidney yang deficiency, effects of hyperlipidemia on theexpression of LXR-α protein as the breakthrough point, study of themechanism of lipid in JAK2/STAT3signal transduction pathway anddeficiency of Kidney Yang high hyperlipidemia rats LXR-α protein and itsregulation and control, and to observe the effect of Yougui Pill on kidney yangdeficiency model rats blood lipid indexes related to. By detecting the contentof its TG, TC, LDL-C and HDL-C, JAK2, STAT3, changes of LXR α,SREBP-1c, FAS,ABCA1and CETP gene mRNA and protein expression, inorder to LXRα expression pathway within the cell to elucidate the mechanismof kidney yang deficiency syndrome in hyperlipemia.Methods: This study was performed at8weeks of age of60SD rats, male,were randomly divided into normal control group, model group and YouguiPill Treatment Group. Except the normal control group, the remaining ratswere intramuscular injection of hydrocortisone method to copy the rat modelof kidney yang deficiency. The rats in the normal control group every morning8:00intramuscular injection of physiological saline1ml/200g weight, the restrats at8:00intramuscular injection of hydrocortisone1ml/200g (drug concentration was5mg/ml), lasted15days; at the sixteenth day, Yougui Pillgroup gavage Yougui Pill suspension2ml/200g (drug concentration was0.243g/ml), the other group of rats physiological saline2ml/200g, which hadbeen lasted for30days. All animal in experiment sixteenth and forty-sixthdays were placed in metabolic cages in the corresponding markers,24hoururine; at the sixteenth day and blood samples were collected from the tailartery,at forty-sixth day to complete the animal with the sampling blood, thencut open the abdominal wall, isolated from rat liver, and associated indicatorsdetection:①detecting24urinary17-OH-CS and serum testosterone (T)content;②the detection of TG, TC content, LDL-C and HDL-C;③changedetection index of the liver, and to observe the change of structure of livertissues;④the immunohistochemical staining method was used to detect theexpression of LXRα protein in liver tissue;⑤the detection expression ofJAK2STAT3, LXRα, SREBP-1c and FAS mRNA in liver tissue of rats andimmunoblot assay for detection of SREBP-1c and FAS；⑥the detectionmRNA expression of ABCA1and CETP in the plasma of rats on the46th day,and immunoblot assay for detection of ABCA1and CETP；Results:①24hours urine and Testosteron: At the sixteenth day,comparingwith the normal control group, the model group and the Yougui Pill group ratswhich of urinary17-OH-CS and T was significant decreased（P<0.01）,butthere was not any significant differences in the model group and the YouguiPill group. At the forty-sixth day,comparing with the normal control group, themodel group was still significant decreased（P<0.01）,but comparing with themodel control group, the Yougui Pill group rats were dramatically increased（P<0.01）.②serum indicators testing: At the sixteenth day, comparing withthe normal control group, the model group and the Yougui Pill group ratswhich of TG、TC、LDL-C were significant increased（P<0.01）, in the contentof HDL-C was significant decreased （P<0.01）.,but there was not any significant differences in the model group and the Yougui Pill group; At theforty-sixth day,comparing with the model control group, the Yougui Pill grouprats in the content of TG、TC、LDL-C were dramatically decreased（P<0.01）,the HDL-C was dramatically increased（P<0.01）;③the liver index testing:compare with the normal control group, the model group and the Yougui Pillgroup rats whice of the liver index was were obviously increased（P<0.05）,compare with the model control group, the Youguiwan group rats wereobviously decreased（P<0.05）; Light microscopy showed: comparison of thenormal control group,the model group rats were significantly disorder in thehepatic lobule structure and liver cell cord fuzzy or even disappear, liver cellmorphological heterogeneity, cell volume increases, cytoplasm rarefaction, thecytoplasm is full of different sizes of lipid droplets, visible part of the nucleusis squeezed to the cell membrane, some nucleus were dissolved.In the portalarea the inflammatory cell was obvious, and focal necrosis were seen scatteredin the other. The structure of hepatic lobule Yougui Pill group rats were intact,liver cells were arranged in order, reduce the size, lipid droplets less than themodel group, fatty degeneration of liver cells is significantly improved,andhad a little inflammatory cell;④the LXR-α protein by immunohistochemistry methods showed: model LXR α protein in liver of rat was strongest,Yougui Pill group, the normal group was weakened.⑤Detection of theexpression of related gene index in the liver: compare with the normal controlgroup, the model group rats whice of JAK2、STAT3、LXRα、SREBP-1c andFAS mRNA expression had dramatically increased（P<0.01）,the Yougui Pillgroup had obviously increased（P<0.05）;but compare with the model controlgroup, the Yougui Pill group rats had dramatically decreased（P<0.01）;SREBP-1c and FAS in Western blotting showed: The maximum value of graywas the model group, which the film with the most coarse, protein expressionwas the strongest, followed by Yougui Pill group, once again into the normal group, the minimum gray value, the film strip the fine and protein expression.⑥Results the mRNA expression of ABCA1and CETP: compared with normalcontrol group, the expression level of ABCA1and CETP in the plasma of therats in the model group was significantly increased in mRNA (P <0.01); theexpression level of ABCA1and CETPmRNA Yougui Pill group increasedsignificantly (P <0.05); but the expression level of Yougui Pill group than inmodel group and ABCA1CETPmRNA significantly decreased (P <0.01).The results showed that the expression of ABCA1and CETP protein inplasma of rats in model group: gray value maximum, the film with the mostcoarse, protein expression was strongest, followed by Yougui Pill group, onceagain into the normal group, the minimum gray value, the film with the mostfine, protein expression.Conclusion:①By intramuscular injection of hydrocortisone replicatedkidney-yang deficiency rat model, general model rats accorded with thediagnostic standard of kidney yang deficiency rat model, and24hour urine17-OH-CS, in the serum TG, TC, LDL-C were increased, HDL-C wasdecreased, which showed that kidney-yang deficiency induced hyperlipidemiarat model was duplicated successfully.②Hyperlipidemia directly lead to disorder lipid metabolism of liver andmorphological changes.③Yougui Pill inhibited JAK2, STAT3, LXR α, SREBP-1c, FAS pathway toreduce triglyceride synthesis.④Yougui Pill was to be down-regulation of JAK2, STAT3, LXRα,ABCA1and CETP to reduce the deposition of cholesterol in plasma cholesterol,promote the metabolism of liver.This is the first study by intramuscular injection of hydrocortisone methodto replicate the success of the kidney-yang deficiency rats model ofhyperlipidemia, using the tonifying kidney drug--Yougui Pill can reduce hyperlipidemia in the deficiency of kidney-yang.The kidney-yang deficiencyinduced to hyperlipidemia model rats by inhibiting of adrenal corticalhormone of large dose of hydrocortisone function, and activating of theJAK2/STAT3pathway, the LXRα expression, and the expression of itsdownstream of SREBP-1c, increased FAS,ABCA1and CETP protein, so thatthe TG and TC content in serum increased significantly. Accordingly, themechanism of Yougui Pill regulating blood lipid metabolism, lipid disorderscorrection may be achieved by inhibiting the overexpression of LXRα protein.