Protective Mechanism Of Astragalus-salvia Miltiorrhiza In Improving Hypertensive Kidney Damage Based On MicroRNA-466b-5P

Objective: In order to analyze the tutor’s medication rules and core drugs,by collecting the tutor’s prescriptions for hypertensive kidney damage.On this basis,mi R-466b-5p was overexpressed in mice,and then the indicators of renal damage in mice were analyzed,and its target was investigated.Afterwards,the intervention of Astragalus-Salvia miltiorrhiza was used to evaluate the indicators of renal damage in mice,and to explore the mechanism of Astragalus-Salvia miltiorrhiza to improve hypertensive renal damage by regulating mi R-466b-5p.Methods: 1.The clinical research adopted the retrospective research method,collected 96 Chinese herbal prescriptions for the treatment of hypertensive kidney damage by the tutor,and analyzed the frequency of medication,siqi,wuwei,and the meridian with the help of the ancient and modern medical record cloud platform software.The SPSS software was used to conduct cluster analysis and association analysis on the drugs,and the core drug pairs were screened out for further animal experimental research.2.The differentially expressed(DE)genes of spontaneously hypertensive rats(SHR)were screened for by mi RNA sequencing in our previous studies.We selected the most significant DE gene to construct recombinant adeno-associated virus(r AAV)to transfect mice.A total of 30 8-week-old C57BL/6 mice were used and 15 mice were randomly selected to be injected with r AAV-mi R-466b-5P in the tail vein to construct a mi R-466b-5p overexpression group,and the remaining 15 mice were injected with AAV-9 vector as a blank control group.After 6 weeks of transfection,the fluorescence in vivo of the mice was observed using an imaging system,The levels of the blood pressure,urine β2-microglobulin(β2-MG),β-N-acetylglucosaminidase(NAG),microalbumin(m ALB)and serum cystatin C(Cys-C),angiotensin II(Ang II),and C-reactive protein(CRP)were measured.Three mice were randomly selected from each group for kidney pathological sections.The predicted target genes and downstream genes were detected by RT-q PCR and Western blot.Six weeks after transfection,we randomly divided into 2 groups(n=6):Group A(Astragalus-Salvia + mi R-466b-5P overexpression group)used astragalus formula granules: 2.036g/kg + salvia formula granules: 0.255g/kg for intragastric administration;Group B(mi R-466b-5P overexpression group)was intragastrically administered with the same volume of normal saline;Group C(Astragalus-Salvia +normal control group)used astragalus formula granules: 2.036g/kg + salvia formula granules: 0.255g/ kg for intragastric administration;Group D(normal control group)was intragastrically administered with the same volume of normal saline.After intragastric administration for 28 days,the urine β2-MG,NAG,m ALB and serum Cys-C,Ang II,CRP and renal pathology were observed.Finally,we determined its target genes by RT-q PCR and Western blot assay.Results:1.Clinical study: The main medicines used by the tutor to treat hypertensive kidney damage were: Astragalus 91 times,Salvia 80 times,Angelica 75 times,Chinese yam 73 times,Chuanxiong 73 times,Fried Atractylodes 73 times,and Taizishen 71 times.There were 579 times in total of 579 medicines classified as siqi.The wuwei were mainly sweet,a total of 959 times.Drugs are classified into the spleen meridian,a total of 841 times.The drugs used by the tutor to treat hypertensive kidney damage are mainly traditional Chinese medicines of invigorating qi and promoting blood circulation.Astragalus-Salvia miltiorrhiza is the tutor’s core drug pair for treating hypertensive renal damage,with a frequency of 77 times,a confidence level of 96%,and a support level of 80%.2.In vivo study: Mi R-466b-5p was found to be highly expressed in spontaneously hypertensive rats(SHR)by sequencing,and we constructed recombinant adeno-associated virus to overexpress mi R-466b-5P in C57BL/6 mice.After 6 weeks,the fluorescence in the mice was observed by the small animal imaging system,verifying that the adeno-associated virus was successfully transfected.We further detected the blood pressure of the two groups of mice,and the blood pressure in the overexpression group was significantly increased(P<0.01).The urine and serum of mice were detected,and the levels of β2-MG,NAG and m ALB in the urine of mice were significantly increased(P<0.01),and the levels of Cys-C,Ang II and CRP in the serum were significantly increased(P<0.01).Pathological changes of hypertensive renal damage such as glomerulosclerosis and renal tubular fibrosis was observed in the kidneys.The expression level of mi R-466b-5p in mouse kidney was detected by q PCR,and mi R-466b-5p in the overexpression group was up-regulated(P< 0.05).The potential functional target gene HAS2 of mi R-466b-5P and the downstream genes of HAS2 were then detected.In the kidneys of mice overexpressing mi RNA-466b-5p,the m RNA level of HAS2 was significantly down-regulated(P<0.01),and the m RNA level of TGFBR1 was significantly up-regulated(P<0.01).HAS2 and TGFBR1 were selected for Western Blot detection,and the results showed that the protein level of HAS2 was decreased(P<0.05),and the protein level of TGFBR1 was decreased(P<0.05).Compared to and group D,urine β2-MG,urine NAG,urine m ALB,serum Cys-C,serum Ang II,serum CRP were significantly increased in group B(P<0.01).Compared with group B,serum Cys-C and serum CRP were significantly increased in group A(P<0.01),urine β2-MG,NAG,m ALB,serum Ang II did not change significantly(P>0.05).Compared with group D,the kidneys in group B had pathological manifestations of hypertensive renal damage,including glomerular sclerosis and tubular fibrosis.Compared with group B,group A has been improved.Compared with group D,hyaluronidase 2(HAS2)was significantly down-regulated in group B,and HAS2 was reversed after astragalus-salvia miltiorrhiza intervention.Conclusions:1.Clinical study: The main medicines used by the tutor to treat hypertension and kidney damage were astragalus,salvia,angelica,yam,chuanxiong,fried Atractylodes,and Taizishen.The siqi of the medicine were mainly ping,and the wuwei were mainly sweet,which are mainly classified into the spleen meridian.Astragalus-salvia was the core drug pair for the tutor’s treatment of hypertensive kidney damage.2.In vivo study: mi R-466b-5P overexpression leads to the significant manifestations of hypertensive renal damage,including increased blood pressure,increased β2-MG,NAG,m ALB in urine,increased Cys-C,Ang II,and CRP in serum,and renal pathological changes in mice.Astragalus-salvia intervention improves the performance of hypertensive kidney damage in mice overexpressing mi R-466b-5P.The renal protection mechanism of Astragalus-Salvia miltiorrhiza pair may relate to down-regulation of mi R-466b-5P and up-regulation of HAS2 expression.