Hispidulin Enhanced Autophagic Flux Inhibits PDGF-stimulated RVSMCs Proliferation By Targeting JAK2/mTOR Signaling Pathway

Background:With the aging of Chinese population gradually deepening and the decrease of the onset age,the incidence of coronary atherosclerotic heart disease is increasing.The treatment of coronary heart disease contains diet prevention,drug control,surgical treatment.Percutaneous coronary intervention(PCI)is still considered to be the best option for coronary atherosclerotic heart disease.One of the complications of PCI is restenosis.With the invention of drug-eluting stents,the incidence of restenosis decreased from about 30%-50%to 10%.However,restenosis after PCI is still one of the complications in long-term survival due to the increasing number of coronary atherosclerotic heart disease.As a flavonoid compound extracted from traditional Chinese herbal medicine,the study showed that hispidulin has strong biological effects of anti-inflammatory,antioxidant,antithrombotic,and anti-proliferation.As an upstream protein of mTOR,I suspect that hispidulin can affect the proliferation of vascular smooth muscle by regulating autophagy.Purpose:1.To investigate whether hispidulin can inhibit stenosis caused by smooth proliferation after vascular balloon injury in rats,and to confirm the inhibitory effect of hispidulin on the proliferation of vascular smooth muscle cells;2.To investigate whether the inhibitory effect of hispidulin-precursor on vascular smooth muscle cells was caused by autophagy;3.Explore the signaling pathway through which high precursors promote autophagy flux.Methods:The experiment is divided into two aspects:cell experiment and animal experiment.To verify the effect of high CVT on balloon injury model and proliferation model of vascular smooth muscle cells in vitro.In the experiment,PDGF-bb was used to stimulate vascular smooth muscle cells to establish a cell proliferation model,and a balloon injury model was established by balloon injury to the common carotid artery of SD rats.The supposition were proved by EDU,CCK-8,Western Blot,HE staining,immunohistochemistry,and projection electron microscopy.NineAlliance was used for Western Blot band analysis,ImageJ analysis for immunofluorescence and sections,SPSS20.0 for data analysis,GraphPad Prism 8.0 for statistical graph,PS 2019,and AI 2019 for the graph.Results:1.The area of carotid artery intima and vascular lumen in rats was observed.The ratio of the vascular lumen in the model group was significantly lower and the ratio of intima was significantly higher than that in the sham group.After 2 weeks of intraperitoneal injection of hispidulin at 40mg/kg,the lumen ratio increased and the intimal ratio decreased in the treatment group compared with the model group(P<0.05).Comparing the weight changes of rats before and after treatment was proved that hispidulin had no inhibitory effect on the growth of rats.2.In the in vitro experiment,We set hispidulin at different concentrations of 20,40,and 60 μmol/L groups.the comparison between groups was conducted through the EDU test,and it was found that hispidulin could inhibit vascular smooth muscle proliferation(P<0.05),CCK-8 test results were consistent with those of EDU test(P<0.05).The expression of proliferative-related,phenotypic-related,autophagy-related proteins was analyzed by Western Blot analysis,and it was found that the expression of PCNA could be down-regulated by hispidulin(P<0.05),up-regulated the expression of α-SMA,SM22-α(P<0.05).in the meantime hispidulin decreased the P62,increased LC3-Ⅱ/LC3-Ⅰ(P<0.05).The results showed that hispidulin inhibited proliferation and phenotypic transformation and promoted the flux of autophagic.3.In the follow-up experiment,it was found that after the treatment of vascular smooth muscle cells with hispidulin and chloroquine,the inhibition effect of hispidulin on the proliferation of vascular smooth muscle cells was reversed in the EDU test,the CCK test and Western Blot(P<0.05).Differences in the number of autophagosomes were observed by confocal microscopy and electron microscopy(P<0.05),which suggests that inhibition of proliferation of vascular smooth muscle cells by hispidulin was achieved by patency of autophagic flux.In the end,we further found that hispidulin inhibits the JAK2/mTOR pathway to promote smooth muscle cell autophagic flux.Conclusions:The experiment verified for the first time that hispidulin regulates autophagy flux by regulating the JAK2/mTOR signaling pathway,thus inhibiting vascular stenosis caused by the proliferation of arterial smooth muscle cells after balloon injury.