Preliminary Research Of The Mechanism Of MiRNA150in Regulating Blood Pressure

As an endogenous uncoded RNA, miRNA regulates gene expressionthrough degradation or translation inhibition. The role of miRNA150inimmune regulation has been known, and its potential role in regulating thecardiovascular system is unclear. This research includes the followingitems:1. To analyze the homeostasis of the cardiovascular system ofmiRNA150knockout mouse, to see if there is myocardial cell hypertrophyor renal impairment;2. To analyze, from a whole level to a molecular level,impairment of endothelium as well as oxidative stress, so as to explore thepossible mechanism for the above manifestations, and to conductshort-term intervention with Eplereone;3. To analyze the possiblepathological and physiological mechanisms that might cause renal damagethrough analyzing the mechanism of age-related renal impairment, so as toprovide clues for further exploration of the regulation of miRNA150inblood pressure and its impairment to renal function. This paper can bedivided into three parts. Part Ⅰ. Homeostasis of the Cardiovascular System of miRNA150-knockout MiceObjective: To research systematically the manifestations of thecardiovascular system of miRNA150knockout mice. Methods: miRNA150knockout mice (KO Group,n=8)and wild-type mice (WT Group,n=8)were compared. Blood pressure was monitored with tail-cuff method; themorphological changes and weight indexes of hearts, kidneys and adrenalglands were observed after16w. The changes of myocardial tissue structureand cardiomycyte cross-sectional area (CSA) were observed with HEstaining. The changes of renal tissue structure were observed withMasson-trichrome staining. The changes of adrenal cortex were observedwith immunofluorescence. The expressions of T cell, B cell and B1cellwere monitored by flow cytometry. Results:(1)The systolic pressure ofmiRNA150-KO mice increased from11w and continued;(2)the mass indexof adrenal glands increased (P<0.05), accompanied with adrenal cortexproliferation;(3)The B1cell content in KO spleen is2.6times of that inWT Group;(4) Compared with WT Group, CSA in KO Group increasedsignificantly;(5) Glomerular and tubular interstitial collagen depositionwas obvious in KO Group. Conclusion: miRNA150-KO mice presentedwith the manifestations of hypertension, which showed proliferation ofspleen B1cells, cardiocytes hypertrophy, adrenal cortex thickening andrenal structural damage. Part Ⅱ Research of the Mechanism of miRNA150in RegulatingBlood PressureSection One CYP11B2Expression and Endothelial FunctionChanges of miRNA150-KO MiceObjective: To preliminarily investigate into the mechanism ofhypertension forming in miRNA150-KO mice through analyzing thechanges of aldosterone synthase CYP11B2and endothelial function.Methods: In20w, plasma of miRNA150knockout mice (KO Group,n=8)and wild-type mice (WT Group,n=8)was taken to determine the plasmaaldosterone level; the CYP11B2expression was detected byimmuno-histochemistry and Western blot; endothelium function in aorticwas detected; the expression of superoxide in situ in aorta, kidney and heartwas detected with DHE staining; the activity of NADPH oxidase in aortawas detected with lucigenin chemiluminescence method. Results:(1) KOGroup had a higher plasma aldosterone level than WT Group (P<0.05);(2)KO Group had a higher level of CYP11B2in both IHC and Western-blotdetection(P＜0.05,P＜0.01);(3) Compared with WT Group, theendothelial diastolic function was impaired (P＜0.05);(4) KO Group hada higher level of superoxide production(P＜0.05), and the activity ofNADPH oxidase was also increased obviously(P＜0.01). Conclusion: The increase of CYP11B2expression and the endothelial dysfunction in aortamight be the mechanism for the high blood pressure of miRNA150-KOmice. Section Two Eplereone Short-term Intervention Experimenton miRNA150-KO MiceObjective: To observe the effect of Eplereone on the blood pressure,aorta endothelial function and the production of oxidative stress, so as tofurther discuss the possible mechanism for the blood pressure ofmiRNA150-KO mice. Methods: miRNA150-KO mice were randomlydivided into DMSO group(KO-DMSO, n=6)and EPL group(KO-EPL,n=6); WT mice were divided into DMSO group (WT-DMSO,n=6) and theEPL group (WT-EPL, n=6). The intervention for EPL group wasintraperitoneal injection50mg/Kg per day for5days.(1) The body massand urine volume were observed;(2) The changes of tissue and organ massindexes and plasma aldosterone were observed;(3) Superoxide productionin kidney and aorta was observed by DHE staining;(4) The activity ofNADPH oxidase was measured;(5) The expression of8-OHDG wasmeasured by IHC;(6) The protective function of EPL group to the kidneywas observed. Results:(1)Compared with KO-DMSO group, KO-EPLgroup showed a decreased blood pressure(P＜0.01);(2) EPL intervention increased the urine volume of KO group.There was no any change ofplasma aldosterone level;(3) EPL improved the endothelial function of KOmice(P＜0.05), and down-regulated the activity of NADPH oxidase(P＜0.05);(4) EPL reduced the production of superoxide in kidney and aorta(P＜0.05,P＜0.01)；(5) EPL reguced the expression of8-OHDG protein.Conclusion: The short-term intervention with Eplereone can decrease theblood pressure of KO mice, inhibit the activity of NADPH oxidase,improve the endothelial function in aorta and reverse renal damage. Part Ⅲ Research of Related Factors Leading to Aging-relatedKidney DamageObjective: Aging is associated with a decline in renal function.Klotho is a recently-discovered anti-aging gene. The purpose of this studywas to determine changes in klotho, endothelin (ET) receptors, andsuperoxide production in kidneys of aged rats. Methods: Twenty aged rats(male,27months) were divided into an Old Impaired group (n=9) and anOld Intact group (n=11) according to a cognitive function test. A group of12month-old rats (n=10) was used as a Young Intact group. Blood wascollected for measuring serum creatinine and ET-1concentration. Renalhistology was evaluated using PAS staining and trichrome staining. The in situ superoxide production was measured in kidneys usingdihydroethidium staining. Klotho protein, ET receptors, Nox2,interleukin-6(IL-6), and MnSOD expression in renal cortex and medullawere assessed by immunohistochemistry and western-blotting. Results:Plasma creatinine was increased significantly in the Old Impaired group,suggesting impaired renal function. Aged rats showed glomerulosclerosisand tubulointerstitialfibrosis. These pathological changes were markedlyaggravated in the old cognitively impaired than in the old cognitively intactanimals. Notably, aged rats demonstrated a significant decrease in klothoprotein expression in renal cortex and medulla. Protein expression of IL-6,Nox2, ETA receptors and superoxide production were increased whereasMnSOD and ETb receptors expression were decreased in kidneys of theaged rats. Interestingly, these changes were more pronounced in the oldimpaired than in the old intact rats. Conclusions: The aging-relatedkidney damage paralleled with the cognitive function impairment. Theaging-related kidney damage was associated with downregulation of klotho,ETB, and MnSOD expression but upregulation of ETA, IL-6, and Nox2expression and superoxide production. These findings raise the necessityto further assess the roles of these factors and their relationship inaging-related kidney damage.