Effect Of Huoxue Qianyang Qutan Recipe On Risk Factors Of Early Renal Damage Induced By Obesity-Related Hypertension And Its Protective Mechanism Via SIRT1/NF-κB Inflammatory Pathway

Objective:Firstly,the clinical efficacy of Huoxue Qianyang Qutan Recipe(HQQR)on risk factors of early renal damage in obesity related hypertension(OBH)was evaluated by randomized controlled trials.Secondly,network pharmacology was used to screen out the core drug components,key targets and signal pathways of HQQR in the intervention of hypertensive renal damage.Finally,based on the core targets and mechanisms of network pharmacology screening and preliminary verification of clinical research,the potential mechanism of HQQR in the intervention of early renal damage in OBH was further explored by using spontaneously hypertensive rats(SHR).The purpose of the study is to provide a multi method(network pharmacology-clinical trials-animal experiments)and multi-level(component-target-pathway)research ideas and theoretical basis for the prevention and treatment of OBH target organ damage with traditional Chinese medicine.Methods:Part 1,Clinical Study:80 patients with early OBH renal damage were randomly divided into treatment group and control group in Yueyang Hospital(from January 2019 to March 2021).The control group was treated with standard western medicine for hypertension,while the treatment group was treated with HQQR on the basis of the control group for 3 months.The clinical data were collected to evaluate the clinical efficacy of HQQR before and after treatment.Part 2,Network Pharmacology Research:Via searching and screening of the network database,we obtained the chemical components of HQQR and the targets of hypertension related renal damage.Using Cytoscape software,we further constructed the interaction network of drug components,targets and signal pathways.Then the hub targets of HQQR were obtained from the components-targets-pathways network.Part 3,Experimental Study:The OBH rats were prepared and accepted drug intervention 10 weeks.The general condition of rats in each group was observed,and the physical and chemical indexes(body weight,Lee index,abdominal circumference,blood pressure,glucose and lipid metabolism,renal function,early renal damage index)were measured.The renal pathology and ultrastructure were observed by HE staining and transmission electron microscope.The serum levels of Ang II and IL-6 were detected by ELISA.qRTPCR and Western blot were used to detect the gene and protein expression of SIRT/NFκB signaling pathway.Result:Part 1,Clinical Study:(1)Baseline:88 patients who met the criteria were included in this study,and 80 patients completed the intervention study.The shedding rate was 9.1%,including 41 patients in the treatment group and 39 patients in the control group.The general patient information(including gender,age,course of hypertension,weight,waist circumference,BMI,SBP,DBP,hypertension classification)of the two groups were balanced and comparable(P>0.05).(2)UACR:Intra group comparison,there was no statistical difference in the UACR of the control group(P>0.05),while the UACR of the test group was statistically different(P<0.01)before and after treatment.Comparison between groups,there was significant difference in UACR after treatment(P<0.05).The total effective rate was 59%in the control group and 80.5%in the experimental group(P<0.05).(3)Effect on blood pressure and ambulatory blood pressure:Intra group comparison,before and after treatment,there were significant differences in systolic and diastolic blood pressure(P<0.05).With medication,the systolic and diastolic blood pressure between the two groups is different(P<0.05).The total effective rate of the experimental group was 75.6%,and that of the control group was 56.4%(P<0.05).Ambulatory blood pressure:A total of 27 patients(14 cases in control group and 13 cases in the treatment group)were monitored by ambulatory blood pressure.24hSBP,nSBP and nDBP in the treatment group had a downward trend before and after treatment(P<0.05)while the P value in the control group was more than 0.05.The results of blood pressure variability showed that the change is not obvious in both treatment group and the control group before and after treatment(P>0.05).(4)Influence of TCM syndrome score:comparison within the groups before and after treatment showed that there was significant difference in TCM syndrome score(P<0.01).After the intervention,the treatment group score was lower than control group score(P<0.01).The effective rate of the treatment group was 82.9%,while the control group is 69.2%(P<0.01).Yang hyperactivity syndrome:the vertigo,irritability,dry mouth,palpitation and insomnia in the treatment group were lower than those in the control group after treatment(P<0.01 or P<0.05).Blood stasis syndrome:the headache symptoms of the treatment group decreased.It is different with control group(P<0.01).Phlegm turbid syndrome:the head weight,dyspnea and chest tightness in the treatment group declined compared with the control group(P<0.01 or P<0.05).(5)Influence of inflammation related risk factors:Compared before and after treatment,the treatment group showed a decrease in IL-6,hs-CRP,MHR and NLR(P<0.01),while the control group showed no significant difference in IL-6,hs-CRP,MHR and NLR(P>0.05).There were significant differences in IL-6,hs-CRP and MHR between the two groups after treatment(P<0.05).(6)Influence on the physique indexes and glucose and lipid metabolism index:Intra group comparison,the weight,waist circumference and height of the treatment group were decreased(P<0.01).The BMI between the treatment group and the control group is different after treatment(P<0.05),The change of body weight and waist circumference was not significant(P>0.05).Glucose and lipid metabolism index:The TG,TC and insulin resistance index of the treatment group were decreased before and after treatment(P<0.01 or P<0.05),while the control group showed no statistical difference(P>0.05).After treatment,the glucose and lipid metabolism between the treatment group and the control group show no obvious change(P>0.05).Part 2,Network Pharmacology Research:(1)Screening of components of HQQR:598 chemical components of HXQYQT recipe were selected,and 35 core drug components were obtained,including tanshinaldehyde,tanshinone,tanshinol,tanshinaldehyde,chuanxiongduo,yangchuan-xiongquinone,rhynchophylline,isorhynchophylline,yumixanthin,stigmasterol,luteolin,jinshengcao,etc.(2)Drug targets of hypertensive renal damage:a total of 4320 targets related to hypertensive renal damage were retrieved.Among them,158 drug disease intersection genes were obtained through screening,which are the targets of HQQR in the intervention of hypertensive renal damage.The core genes include VEGFA,JUN,IL6,PTGS2,AKT1,CXCL8,CASP3,MAPK8,MMP9,STAT3,MAPK1,IL10,CCL2,FOS,MYC,EGF,IL4,MAPK14,IL1B,IFNG.(3)Enrichment analysis of GO and KEGG:The main gene functions of HQQR in treating hypertensive renal damage are anti-inflammatory response and anti oxidative stress response.Combined with KEGG enrichment analysis and cytohubba screening results,NF-κB signaling pathway plays an important role in the treatment of hypertensive renal damage with HQQR.Part 3,Experimental Study:(1)Comparison of general condition,weight and abdominal circumference:Before intervention,OBH rats model group showed abdominal obesity,dark and fluffy fur,decreased urine volume,dark color,loose stool,unclean perianal,aggressive and irritable.After the intervention,the above phenomena were improved,and the fighting was significantly reduced.After dissection,it was found that the content of perirenal fat in OBH group was significantly higher than that in SHR normal diet group.The body weight and waist circumference of OBH rats were higher than those in SHR group(P<0.01).After the intervention,the increasing trend of body weight in the OBH HQQR group was significantly slower than that in the OBH model group,and the body weight in the OBH HQQR group was lower than that in the same age OBH model group from 8 weeks after intervention(P<0.05).The abdominal circumference in the OBH HQQR group and the western medicine group was declined compared with the OBH model group.The abdominal circumference in the OBH HQQR group was lower than that in the same age OBH model group from 6 weeks after intervention(P<0.05 or<0.01).(2)Comparison of blood pressure:SBP and DBP of OBH group were higher than those of WKY group and SHR common food group(P<0.05 or P<0.01);After medication,SBP and DBP of OBH HQQR group and western medicine groups were significantly lower than those of OBH model group.The SBP and DBP in the western medicine and HQQR groups were lower than those in the same age OBH group(P<0.01),and HQQR could control blood pressure more stably in a time-dependent manner.(3)Comparison of Ang Ⅱ and IL-6 indexes:After intervention,the serum Ang Ⅱ content in the western medicine group and the HQQR group was lower than that in the OBH model group(P<0.01).Compared with the OBH model group,the serum IL-6 levels in the western medicine group and the HQQR group were significantly lower(P<0.01).(4)Early renal damage index:After intervention,compared with the OBH model group,the HQQR group can reduce the renal damage index mAlb,UCR,NAG,Cys-C(P<0.01 or P<0.05),β2-MG and Urea had a downward trend,while did not show statistical significance.Western medicine group could reduce β2-MG,Urea and mAlb(P<0.01 or P<0.05),but had no significant intervention on UCR,NAG and Cys-C.(5)Pathological morphology and ultrastructure of kidney:After intervention,compared with the model group,western medicine group and HQQR group,the pathological morphology of kidney was improved,the infiltration of inflammatory cells was reduced,and the ultrastructure of kidney was improved.(6)Comparison of glucose and lipid metabolism indexes:After intervention,FBG and HOMA-IR of rats in western medicine group and HQQR group were lower than those in OBH group(P<0.01).After 10 weeks of intervention,TG,TC and LDL-C in the western medicine group and the HQQR group were declined opposed to the OBH model group(P<0.05),and HDL-C was higher than that in the OBH model group(P<0.05).(7)SIRT1/NF-κB gene and protein expression:SIRT1 expression:After intervention,compared with the WKY group,the SIRT1 gene and protein expression levels in the OBH model group were significantly decreased(P<0.001).Compared with the OBH model group,the expression levels of SIRT1 gene and protein in the HQQR group and the western medicine group recovered and increased,and the difference was statistically significant(P<0.01).Acetylated NF-κB expression:Compared with the WKY normal group,the acetylated NF-κB protein expression level of the OBH model group was significantly higher than that of the WKY normal group(P<0.001).Compared with the OBH model group,the expression level of acetylated NF-κB protein in the HQQR group and the western medicine group decreased(P<0.01).IL-6 expression:Compared with the WKY normal group,the IL-6 gene and protein expression level of the OBH model group was significantly higher than that of the WKY normal group(P<0.001).Compared with the OBH model group,the levels of IL-6 gene and protein expression in the HQQR and the western medicine group decreased,and the difference was statistically significant(P<0.05).Conclusion:(1)Cooperating with western medicine HQQR can be applied to reduce blood pressure,improve early renal damage,correct obesity,improve glucose and lipid metabolism,inhibit inflammatory risk factors,reduce TCM syndrome score and improve clinical symptoms.(2)Based on the network pharmacology,salvia miltiorrhiza is the main component of HQQR,which indicates that Junyao plays an important role in the prescription.HQQR may play a similar role as anti-inflammatory factor by regulating the core target of IL-6 and NF-κB signaling pathway in protecting OBH target organs.(3)HQQR can protect OBH early renal damage via up regulating SIRT1 expression,reducing the expression level of NF-κB deacetylation,reducing the release of IL-6 and inhibiting inflammatory reaction.