Mechanism Of Puerarin Regulating Spontaneously Hypertensive Rat Aortic Diastolic Function Through TRPC3

Background:The incidence of hypertension is high and the pathological mechanism is complicated.Vasodilation dysfunction is one of the important pathological foundations of its development.Previous studies have found that puerarin can reduce blood pressure and myocardial oxygen consumption.Our previous studies have confirmed that puerarin can improve the microvascular diastolic function of spontaneously hypertensive rats（SHR）.Little research and controversy.The role of TRPC3 as a member of the TRP family in cardiovascular disease has received widespread attention.At present,it has been found that the opening of TRPC3 channels can promote Ca²⁺influx in vascular smooth muscle cells,thereby regulating the phenotypic transformation of vascular smooth muscle cells（VSMC）and vasomotor effects.VSMC phenotypic transformation is a key factor in regulating vasomotor function.Some scholars have found that Ang II-induced VSMC phenotypic transformation is related to activation of NF-κB and up-regulation of Cx43 expression.Objective:To explore the specific mechanism of puerarin in regulating vasomotor function and smooth muscle phenotype transformation through TRPC3.Methods:Thirty SHRs were randomly divided into hypertension group,telmisartan group,puerarin 40mg/kg^-1group,puerarin mg/kg^-1group and compatible administration group,6 in each group;6 WKY were used as normal control group.One week after the adaptive feeding,the telmisartan group was administered with gavage at mg/kg^-1daily,and the puerarin mg/kg^-1group and 80mg/kg^-1group were administered with mg/kg^-1and mg/kg^-1Intraperitoneal injection,the compatible drug delivery group was intragastrically administered with mg/kg^-1daily and intraperitoneal injection of puerarin mg/kg^-1.Hypertension group and normal control group were injected with equal volume of normal saline daily.Blood pressure and echocardiogram measurements were performed at 0,4,8 and 12 weeks after administration.After the end of 12 weeks of administration,the serum was taken to measure the Ang II content.The thoracic aorta of rats was isolated for HE staining and Masson staining,and the morphological and structural changes of the thoracic aorta were observed under a microscope.Rat thoracic aorta was isolated for immunofluorescence staining,and the expression of TRPC3 in vascular smooth muscle of thoracic aorta in each group was observed under a microscope.At the same time,vascular tension experiments were performed on isolated thoracic aorta vascular rings of each group of rats.The non-specific TRPC channel blocker SKF-96365（5μmol/L）and the specific TRPC3 channel blocker Pyr3（20μmol/L）were used for observation.Is the vasodilation effect of puerarin related to TRPC3?Primary VSMCs were cultured by adherence to tissue blocks and purchased A7r5 cell line.Add primary VSMC and A7r5 to Ang II(10^-7mol/L),Pue(10^-4mol/L)and Ang II(10^-7mol/L)+Pue(10^-4mol/L)and incubate for 24h,using Western Bolt was used to detect the expression ofα-SMA,TRPC3 and Cx43 in primary VSMC and A7r5,and the expressions of TRPC3,Cx43 and NF-κB p65 were observed by immunofluorescence.Results:Puerarin long-term administration can significantly reduce blood pressure in spontaneously hypertensive rats;ultrasound results show that long-term administration of puerarin narrows the lumen diameter of the SHR thoracic aorta;ELISA results show that long-term administration of puerarin reduces SHR serum Ang II Content;HE staining and Masson staining showed that puerarin can significantly thin the thoracic aorta vascular wall of SHR,arrange smooth muscle cells more regularly,and reduce the degree of vascular collagen fibrosis.The expression of TRPC3 was detected by immunofluorescence.The results suggest that puerarin can down-regulate the expression of TRPC3 in the thoracic aorta of SHR rats.In vitro vascular ring experiments,the non-specific TRPC channel blocker SKF-96365（5μmol/L）or the specific TRPC3 channel blocker Pyr3（20μmol/L）were added.The results suggest that puerarin can improve vasodilation function;May be related to TRPC3 on vascular smooth muscle.This indicates that puerarin may regulate the vasomotor function by down-regulating the expression of TRPC3;（not discussed in the results）.Western Bolt results show that puerarin can significantly inhibit the Ang II-induced contraction phenotypic markerα-SMA in primary VSMC and A7r5 Down-regulation of expression indicates that puerarin can inhibit Ang II-induced VSMC phenotypic transformation（not discussed in the results）.Western Bolt and cellular immunofluorescence results showed that Ang II induced the up-regulation of TRPC3 expression in primary VSMC and A7r5,and caused the up-regulation of Cx43 expression.Puerarin could significantly inhibit the up-regulation of TRPC3 and Cx43 expression induced by Ang II.The above results suggest that the regulation of Cx43 expression by puerarin is related to TRPC3.The results of cellular immunofluorescence showed that Ang II induced an increase in nuclear translocation level of NF-κB p65 in primary VSMC and A7r5,and the level of nuclear translocation of NF-κB p65 decreased when puerarin was added.Conclusion:Puerarin inhibits NF-κB activation through TRPC3,thereby inhibiting the expression of Cx43,promoting the conversion of vascular smooth muscle cells to the contractile phenotype,and thereby improving vasomotor function.