Based On The NLRP3 Inflammasome To Study The Antihypertensive Vascular Endothelial Damage Mechanism Of Compound Uncaria Uncaria Tablets

Objective:1.To explain the pathogenesis characteristics and treatment of（Traditional Chinese Medical）TCM of hypertension,to clarify the theoretical of Compound Gouteng Antihypertensive Tablet（CGAT）prescription basis on TCM.To explore mechanism of its multi-component-multi-target-multi-pathway treatment of hypertension,and provide theoretical basis for further experimental research.2.To elucidate the protective effect of CGAT on hypertensive vascular endothelial injury,to study the mechanism of its antihypertensive vascular endothelial injury from the NLRP3 inflammasome pathway,and provide experimental basis for CGAT treatment of hypertension.Methods:1.To summarize and analyze the TCM disease name,etiology,pathogenesis,disease location and syndrome classification of hypertension from 78 journal literatures of national famous old Chinese medicine in recent ten years.Combined with literature investigation and previous clinical research,the pathogenesis of hypertension was put forward,and its treatment and medication were clarified.Used integrated pharmacology platform to build CGAT’s"component target-disease target"network and"TCM-component-target-pathway"action network to explore CGAT’s biological processes that may be involved in the treatment of hypertension.2.30 SD rats of grade SPF were randomly divided into normal group and model group.An intraperitoneal injection of L-NNA（15 mg/kg）for 21 days to establish a rat model of endothelial injury type hypertension.And then the model group was randomly divided into model group,western medicine group（7.615 mg/kg）and TCM group（22.845 g/kg）after successful model replication,successive administration for4 weeks.The SBP and DBP of rats in each group were measured by caudal artery pressure measurement at 1 h after morning gavage of before and after 1,2,3,4 weeks administration respectively.After materials taken,the serum NO、ET-1、IL-1β、IL-18contents were detected by ELISA method.Observed the pathological morphological changes of thoracic aorta by HE staining method.The positive expression of e NOS,ET-1,NLRP3,ASC,Caspase-l in thoracic aorta were detected by immunohistochemistry.IL-1β,IL-18 protein expression of thoracic aorta were detected by Western Blot.The m RNA expression of thoracic aortic NLRP3,ASC,Caspase-l were detected by q PCR.3.The cultured HUVEC cells were randomly divided into normal group,model group,control group,TCM group,inhibitor group and inhibitor TCM group after screening the appropriate intervention concentration of H₂O₂,MCC950 and drug-containing serum by CCK-8 method.The morphological changes of HUVEC cells in each group were observed by inverted microscope after 24 h of serum was added.The cell activity of each group was detected by CCK-8 method.The content change of NO in HUVEC cell culture supernatant was detected by nitrate reductase method,and the content change of ET-1,IL-1β,IL-18 in culture supernatant was detected by Elisa method.The protein expression levels of NLRP3,ASC,Caspase-l in HUVEC cells were detected by Western Blot method.Results:1.Results of literature study showed,there were 19 kinds of TCM diseases of hypertension,most commonly known as vertigo.There were internal and external causes,the internal causes were mainly in endowment deficiency,old body deficiency and internal injury deficiency damage.The disorder of Yin and Yang of liver and kidney was the basic pathogenesis characteristic of hypertension,the disease location was mainly in liver and kidney,the hyperactivity type of liver Yang was the most syndrome type of 65 types.The essential hypertension disease was the syndrome of"asthenia in origin and asthenia in superficiality",which was based on liver and kidney Yin deficiency as the basis,liver Yang hyperactivity,blood stasis blocking collaterals as the standard,with the characteristics of the pathogenesis of"deficiency,blood stasis,wind",treatment should treat deficiency,blood stasis and wind combined.Clinical Medicine based on the traditional efficacy of TCM and the modern pharmacological action of drugs.The treatment effect of CGAT was remarkable by the prescription of nourishing yin and softening liver,removing blood stasis and clearing collaterals.Target network feature analysis showed that,the 36 active components in the predicted CGAT strongly interact with 29 major proteins,such as ATP synthase（ATP1A1、ATP5A1、ATP5C1、ATP5B）and prostaglandin endogenous peroxidase（PTGS1、PTGS2）.Network enrichment analysis showed that,it was involved in the regulation of different links in the pathological process of hypertension through 15 pathways such as adrenergic signaling in cardiomyocytes,calcium signaling pathway,vascular smooth muscle contraction.2.Results of animal experiments showed,compared with the normal group,the caudal artery SBP and DBP were significantly increased in the model group（P<0.01）before and after administration in 1,2,3 and 4 weeks.In the model group,the thoracic aortic intima structure was disordered,the endothelium was incomplete,there were protrusions and defects,the middle membrane was thickened,and the boundary between the outer membrane and the middle membrane was not good.The serum NO content of the model group decreased significantly（P<0.01）,and the ET-1,IL-1β,IL-18 content increased significantly（P<0.01）.The immunoreactive expression of e NOS in thoracic aorta was significantly decreased in the model group（P<0.01）,and the ET-1,NLRP3,ASC,Caspase-l immunoreactive expression were significantly increased（P<0.01）.The protein expression of IL-1β,IL-18 in thoracic aorta significantly increased in the model group（P<0.01）,and the m RNA expression of thoracic aortic NLRP3,ASC,Caspase-l were also significantly increased（P<0.01）.Compared with the model group,the SBP,DBP of caudal artery were significantly decreased in rats after 1,2,3 and 4 weeks after administration（P<0.01）.The intima structure of the aorta in TCM group was improved,the endothelium was intact,the intima was smooth,and the boundary between the outer membrane and the middle membrane was clear.The serum NO content of rats in TCM group increased（P<0.01）,and ET-1,IL-1β,IL-18 content of rats were all decreased（P<0.01）.In TCM group,the positive expression of e NOS in thoracic aorta was increased（P<0.05）,and the positive expression of ET-1,NLRP3,ASC,Caspase-l were all decreased（P<0.05or P<0.01）.IL-1β,IL-18 protein expression of thoracic aorta were decreased in TCM group（P<0.01）,and the m RNA expression of thoracic aortic NLRP3,ASC,Caspase-l in TCM group were decreased（P<0.01）.3.Results of cell experiments showed,The H₂O₂ of 0.2 m M is suitable for establishing HUVEC cell oxidative stress injury model.The MCC950 of 10μM and10%of the drug-containing serum were suitable experimental intervention concentrations.Compared with the normal group,the number of HUVEC cells in the model group decreased significantly,the cells were connected to each other less,the arrangement was disordered,the size distribution was uneven,the cells crumpled and rounded,the cell body and nucleus became smaller,the cell membrane was not smooth,the intracellular structure was blurred,and the phenomenon of shedding was obvious.The survival rate of HUVEC cells in model group decreased significantly（P<0.01）.The NO,ET-1,IL-1β,IL-18 contents of HUVEC cells in the model group were significantly increased（P<0.01）,and the protein expression of NLRP3,ASC,Caspase-l was significantly increased in the model group（P<0.01）.Compared with the model group,the number of HUVEC cells in the TCM group increased significantly,the cell morphology improved,the arrangement was more orderly,most of the cells were polygonal or fusiform,the cell connections were tight,and the cell boundary was clear.The survival rate of HUVEC cells in the TCM group was significantly increased（P<0.01）.The NO,ET-1,IL-1β,IL-18 contents of HUVEC cell secretion in the TCM group were significantly decreased（P<0.01）,and the protein expression of NLRP3,ASC,Caspase-l in the cells of TCM group was significantly decreased（P<0.01）.The protein expression of NLRP3,ASC,Caspase-1in the cells of the inhibitor TCM group was more obvious than that in the TCM group（P<0.01）.Conclusion:1.Based on the characteristics of the pathogenesis of vertigo"deficiency,blood stasis,wind"and the combination of treatment of deficiency,blood stasis and wind.The theory of CGAT prescription is sufficient.This prescription for hypertension has the characteristics of multi-component-multi-target-multi-pathway regulation.2.CGAT can correct the balance disorder of vasoactive substance NO、ET-1 in hypertensive rat model caused by L-NNA.It has good antihypertensive and vascular endothelial protective effects.3.CGAT may achieve protective effects on vascular endothelial injury in a rat model of hypertension by inhibiting the expression of N LRP3、ASC、Caspase-l、IL-1β、IL-18 in the NLRP3 inflammasome pathway.4.CGAT drug-containing serum may play an anti-inflammatory role by inhibiting NLRP3 inflammasome pathway to achieve protective effect on H₂O₂ induced HUVEC cell injury.