The Mechanism Of Aldosterone Inducing Cardiomyocyte Pyroptosis And Angiogenesis In Cardiorenal Syndrome And The Protection Of Traditional Chinese Medicine

Chronic renal heart syndrome is a common complication of chronic kidney disease,in which pyroptosis plays an important role.Therefore,studying its pathogenesis is of great significance for clinical treatment.This study is based on the "holistic concept" of traditional Chinese medicine,using UUO experimental animal model of chronic kidney injury,observing the effects of aldosterone-induced pyroptosis and angiogenesis stimulation on cardiac function and structure,and giving the mineralocorticoid receptor blocker and Yiqi Huoxue Jiedu herbs,to observe the effect of inhibiting pyroptosis,and abnormal angiogenesis,reducing the heart damage of obstructive nephropathy,and provide a certain basis for clinical treatment.Part I: Changes in the structure and function of the heart in rats with obstructive nephropathy and the protective effect of traditional Chinese medicineObjective: To observe structural and functional changes in the heart of UUO rats and the protective effect of Yiqi Huoxue Jiedu herbs.Methods: Male Wistar rats were randomly divided into 4 groups: control group(Sham group),model group(UUO group),eplerenone group(MRB group)and traditional Chinese medicine group(TCM group).No ligation was performed for the rats in the control group while the left ureter is ligated for the rats in other groups.Eplerenone and Yiqi Huoxue Jiedu herbs were administrated in the MRB group and the TCM group respectively for 6 months starting from the first day after surgery.Heart tissues were taken for examination.The heart and kidney function and blood pressure of rats were measured.HE staining was performed to observe the pathology of myocardial tissue.The degree of myocardial tissue fibrosis was observed by Masson and Sirius red staining.The above data were shown as mean ± standard deviation,analyzed by one-way ANOVA and LSD test,with P<0.05 as the standard of significant difference.Results:1.Body weight,heart weight and heart / body specific gravity of rats in UUO ratsCompared with the Sham group,the heart weight of UUO group was significantly increased(P <0.001),and the heart weight of MRB and TCM group was reduced(P<0.05)compared to UUO group;calculating the heart /body ratio,UUO group and EPL group had an increased ratio compared with Sham group(P<0.05),while TCM group had no statistical difference compared with UUO group and Sham group.2.The changes in systolic blood pressure of UUO ratsThere was no significant difference in systolic blood pressure between rats from different groups at 1 and 2 month,but at 3 to 6 months after surgery,the systolic blood pressure in UUO group was higher than that of Sham group(P<0.05).Compared with MRB/TCM group and UUO group,the systolic blood pressure was reduced(P <0.05).3.The renal function of UUO ratsCompared with Sham group,the serum creatinine levels of rats in UUO group were significantly higher(P <0.001).There was no significant difference MRB group and TCM group compared with the UUO group.Concerning the serum urea nitrogen levels,UUO group was significantly higher Sham group(P<0.001).4.The cardiac function of the UUO ratsEF and FS values were significantly lower in UUO group than Sham group(P<0.01),while MRB and TCM groups were significantly higher than UUO group(P <0.001).5.The cardiac tissue pathological changes in UUO ratsHE staining showed that rats in Sham group had complete myocardial structure,with regular morphology,and there was a small amount of infiltration of inflammatory cells between the myocardium and perivascular.In UUO group,myocardial structure of rats was partially incomplete with the characteristic of cardiac myocyte hypertrophy,irregular nucleus,and uniform staining.There was more inflammatory cell between the myocardium,especially around the blood vessels.Compared to UUO group,the inter-cardiac and perivascular inflammatory cells was reduced in MRB and TCM groups.Masson staining and Sirius Red staining showed that a small amount of fibrous tissue was deposited between the myocardium and blood vessels in Sham group rats.In UUO group,there were a large amount of collagen fibers between myocardial tissues,as well as some areas around blood vessels.In comparison,collagen fiber deposition was significantly reduced in MRB and TCM groups.Conclusion: The rat model of unilateral ureteral obstruction showed a decline in renal function at 6 months after operation,and successfully recapitulated the CKD model.UUO rats had adverse effects on myocardial structure and cardiac function.Eplerenone,a MR receptor blocker,and yiqi huoxue jiedu herbs can protect the myocardium of UUO rats and delays the progression of CRS.Part Ⅱ: Study on the mechanism of aldosterone mediated cardiomyocytes pyroptosis to deteriorate cardiac structure and function in UUO rats.Objective: To explore the mechanism of aldosterone-mediated pyroptosis in the occurrence and progression of CRS-4.Methods: Male Wistar rats were randomly divided into 4 groups: control group(Sham group),model group(UUO group),eplerenone group(MRB group)and traditional Chinese medicine group(TCM group).No ligation was performed for the rats in the control group while the left ureter is ligated for the rats in other groups.Eplerenone and Yiqi Huoxue Jiedu herbs were administrated in the MRB group and the TCM group respectively for 6 months starting from the first day after surgery.The serum aldosterone levels was measured;The cardiomyocyte DNA damage was observed by TUNEL.pyroptosis marker GSDMD was observed by immunohistochemical staining;The expression of signal pathway-related protein GSDMD,NLRP3,SGK-1,NF-κB,caspase-1,IL-1β were tested by western blot.RT-PCR was used to detect the m RNA levels of SGK-1 and NF-κB.Results:1.Comparison of serum aldosterone levelsCompared with Sham group,serum aldosterone levels in UUO group were significantly higher(P<0.001);MRB group were significantly lower than UUO group(P<0.001);Compared with the UUO group,MRB group also decreased to a certain extent(P<0.05).2.The DNA damage of cardiomyocytesThe TUNEL positive cells in UUO group were significantly higher than Sham group,and a large number of positive cells appeared in the cardiomyocytes,vascular endothelial cells and perivascular stromal cells.Compared with the UUO group,the number of positive cells in MRB and TCM group was significantly reduced and scattered in a small amount.3.The expression of GSDMD in myocardial tissueThe GSDMD positive cells in UUO group were significantly increased,mainly in cardiomyocytes and interstitial cells.MRB and TCM groups was significantly reduced compared to UUO group.4.The expression of signal pathway-related protein GSDMD,NLRP3,SGK-1,NF-κB,caspase-1 and IL-1βThere was no significant difference in GSDMD-full in myocardial tissue of each group(P>0.05);compared with Sham group,GSDMD-N expression in UUO group was significantly increased(P<0.001),Compared with the UUO group,the expression levels of GSDMD-N in MRB and TCM groups were significantly reduced(P<0.001,P<0.05).Compared with Sham group,The expression of pro-Caspase-1 and Caspase-1 cleavage in UUO group was significantly increased(P<0.001);MRB and TCM groups were reduced compared with the UUO group(P<0.05).Compared with Sham group,the expressions of pro-IL-1β and IL-1βcleavage in UUO group were significantly increased(P<0.001),while the expression levels of MRB and TCM groups were reduced compared to the UUO group(P<0.05).The expressions of NLRP3 and NF-κb in UUO group were significantly higher than Sham group(P<0.001);MRB group were significantly reduced compared to UUO group(P<0.001);TCM group was no significantly different from UUO group.The expression of SGK-1 in the UUO group was significantly higher than that in Sham group(P<0.001);MRB group was significantly reduced compared to UUO group(P <0.01);TCM group was no significantly different from the UUO group.5.The m RNA level of SGK-1,NF-κB,NLRP3 and IL-1βCompared with the Sham group,the SGK-1,NF-κB,NLRP3 and IL-1βm RNA levels were enhanced in the UUO group(P <0.001),however,the expression of those genes in the MRB and TCM groups were decreased obviously(P <0.001).Conclusion: Aldosterone can cause myocardial fibrosis by inducing cardiomyocytes pyroptosis.Aldosterone induced pyroptosis is mediated by the MR/SGK-1/NLRP3 signal transduction pathway.Eplerenone can effectively reduce cardiomyocytes pyroptosis by inhibiting the activation of MR,and play a certain protective effect on the CRS-4.Part III: The role of angiogenesis and endothelial mesenchymal transformation in cardiorenal syndrome and the protection of traditional Chinese medicine.Objective: To investigate the role of endothelial mesenchymal transformation and angiogenesis in cardiac fibrosis of UUO rats.Methods: Male Wistar rats were randomly divided into 4 groups: control group(Sham group),model group(UUO group),eplerenone group(MRB group)and traditional Chinese medicine group(TCM group).No ligation was performed for the rats in the control group while the left ureter is ligated for the rats in other groups.Eplerenone and Yiqi Huoxue Jiedu herbs were administrated in the MRB group and the TCM group respectively for 6 months starting from the first day after surgery.Immunohistochemical staining was applied to detect the expression of CD34 and CD105 in myocardial tissue,and immunofluorescent staining was performed to examine the expression of CD34,COL Ⅰ/COL Ш,SMA,and vimentin.Western blot and RT-PCR were used to detect the expression of VEGFA and VEGFC.RT-PCR was used to detect the level of COLI m RNA.Results:1.The expression of COLI and COLIIIThe expression of COL Ⅰ and COLIII in myocardial tissue of UUO group was significantly higher than that of Sham group,especially in most areas of myocardial interstitial;MRB and TCM group compared with UUO group,the expression of COL Ⅰ and COLIII decreased.2.The expression of CD34 and CD105Immunohistochemical results showed that compared with Sham group,the expression of CD34/CD105 in myocardial tissue of UUO group was significantly increased,while the expression of MRB and TCM group was reduced.Immunofluorescence results showed the same results.3.The co-expression of CD34 and α-SMA / vimentinCompared with the Sham group,more co-expression of CD34 and α-SMA/ vimentin were observed in UUO group,and MRB and TCM groups were reduced compared with the UUO group4.The co-expression of CD34,α-SMA and COL I /COL IIICompared with the Sham group,UUO group showed more co-expression of CD34,α-SMA and COL I /COL III,while MRB and TCM groups were reduced compared with the UUO group.5.The differences expression of CD34 and CD105The expression of CD105 in myocardial tissue is significantly less than that of CD34,but it is more expressed in UUO group than Sham group.In MRB and TCM groups,the expression is somewhat higher than the UUO group.6.The co-expression of CD105 and α-SMA / vimentinCompared with the Sham group,more co-expression of CD105 and α-SMA / vimentin were observed in UUO group,and MRB and TCM groups were reduced compared with the UUO group7.The expression of VEGFA and VEGFC proteinThe expressions of VEGFA and VEGFC in the UUO group were significantly higher than those in the Sham group(P<0.01),while the expressions in the MRB and TCM groups were reduced(P <0.05).8.The m RNA level of COL ICompared with the Sham group,the COL I m RNA levels were enhanced in the UUO group(P <0.001),while the expression of those genes in the MRB and TCM groups were decreased obviously(P <0.01)Conclusion: Angiogenesis and endothelial cell mesenchymal transformation are involved in the formation of myocardial fibrosis in UUO rats.VEGF participates in the regulation of angiogenesis in UUO rats.Aldosterone receptor blocker eplerenone and Yiqi Huoxue Jiedu herbs may reduce cardiac fibrosis by inhibiting angiogenesis and endothelial phenotype transformation.General conclusions:1.The rat model of unilateral ureteral obstruction showed a decline in renal function at 6 months after operation,and successfully replicated the CKD model.2.The UUO rats showed adverse effects in myocardial structure and function.3.Pyroptosis plays an important role in the progress of CRS,which is mediated by aldosterone through activating MR/SGK-1/NLRP3 signal transduction pathway.4.Angiogenesis and endothelial mesenchymal transformation play an important role in the establishment and development of cardiac fibrosis in UUO rats.5.Eplerenone,a MR receptor blocker,and yiqi huoxue jiedu herbs can protect the myocardium of UUO rats and delays the progression of CRS through inhibiting MR activation,pyroptosis,angiogenesis and endothelial phenotype transformation.