| Background and Objective:Cardiovascular and cerebrovascular disease is the main cause of death worldwide.In recent years,with the continuous updating of medical devices and the improvement of surgical techniques of clinical intervention doctors,interventional therapy for cardiovascular and cerebrovascular diseases has became an important treatment.In the past 20 years,although the rapid development of percutaneous coronary intervention has been achieved,the incidence of in-stent restenosis(restenosis,RS)is still as high as 10%,which seriously affects the surgical outcome.Therefore,it is great significance that to explore the pathogenesis and treatment of RS.Studies have shown that proliferation and migration of vascular smooth muscle cells(VSMCs)and inflammatory responses play an important role in vascular restenosis after vascular endothelial injury.Cantharidin(CA)is the main active ingredient of traditional Chinese medicine spotted mites,and it has significant inhibitory effect on the proliferation and invasion of various cancer cells including lung cancer,breast cancer and melanoma,and it has been found to play a role in the inhibition of nuclear factor kappa B(NF-κB)signaling pathway-mediated inflammatory responses in melanoma cells.Therefore,we speculated that cantharidin may also inhibited the proliferation and migration of VSMCs and inhibited the inflammatory response in vascular restenosis after endovascular injury.In order to verify the above speculation,the study was conducted in two parts.In the first part,VSMCs were cultured,and their proliferation and migration were induced by lipopolysaccharide(LPS).The effects of cantharidin on the proliferation and migration of VSMCs and the effect on NF-κB signaling pathway were observed.In the second part,a restenosis model was established by carotid artery balloon injury in SD rats to observe the effect of cantharidin treatment on intimal hyperplasia.Method:1.VSMCs were isolated from thoracic aorta of male Sprague-Dawley rats by tissue adherent method,and the cells were identified by immunofluorescence staining with vascular smooth muscle cell phenotype marker(α-SM-actin)antibody.The proliferation and migration of VSMCs were induced by 1 mg/L lipopolysaccharide(LPS),and observed the effects of different concentrations of cantharidin on VSMCs.The viability of VSMCs and the selection of the concentration of LPS was determinedby cell counting kit-8(CCK-8)assay.Cell proliferation was detected by flow cytometry and CCK-8 assay.Cell migration was detected by transwell assay and wound-healing assay.Total NF-κB p65(NF-κB p65),phosphorylated NF-κB p65(P-p65),IκB-α expression levels were detected by Western blot.The expression levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and Monocyte chemoattractant protein-1(MCP-1)mRNA were detected by real-time quantitative PCR(Q-PCR).The contents of TNF-α and IL-6 in cell culture supernatant were detected by enzyme-linked immunosorbent assay(ELISA).2.Rat carotid balloon injury model was established by Fogarty(2F)balloon catheter.SD rats were randomly divided into sham operation group,injury group and cantharidin(2mg/kg)group.Each group started intraperitoneal injection 1 week before modeling.And continuous administration for 3 weeks,and blood was taken 14 days after operation to detect the levels of TNF-α and IL-6,and the injured carotid artery was taken for HE staining and immunohistochemistry.The expression of NF-κB p65,TNF-α and IL-6 was detected by staining.Results:1.Cell experiment: CCK-8 assay showed that the activity of VSMCs was not affected when the concentration of cantharidin was 0-10 μmol/L.The proliferation of VSMCs was significantly increased when LPS was used at 1 mg/L.The results of CCK-8 assay for cell proliferation activity showed that the proliferation activity of VSMCs was significantly increased after LPS stimulation,and the proliferation activity decreased with the increase of cantharidin concentration(P<0.05).The results of flow cytometry showed that the proportion of S phase cells in VSMCs was significantly decreased after LPS stimulation,while the proportion of cells in G2 phase was significantly increased(P<0.01).However,compared with LPS stimulation group,after stimulation with cantharidin,the number of cells in S phase was significantly increased(P<0.01),and the proportion of cells in G2 phase was significantly decreased(P<0.05).Wound healing assay showed that the migration rate of VSMCs increased by 2.8-fold after LPS stimulation,compared with the control group(P<0.01).But after treatment with cantharidin,the cell migration rate decreased.Transwell results showed that compared with the control group,the number of cells penetrating the chamber after LPS stimulation increased by 3.25 times(P<0.01);and the number of cells penetrating the chamber after treatment with 2.5,5,10 μmol/L cantharidin was decreased by 20.8%,45.2% and 74.8% respectively,compared with the LPS group(P<0.05).Western blot analysis showed that after LPS stimulation,theexpression of IκB-α was significantly decreased compared with the control group(P<0.01).The expression of P-p65 was significantly increased after LPS treatment(P<0.05),while cantharidin inhibited the above effects of LPS in a concentration-dependent manner(P<0.05).Q-PCR results showed that the levels of TNF-α,IL-6 and MCP-1 mRNA in the cells were significantly increased after LPS induction,compared with the control group(P<0.05).Compared with the LPS group,TNF-α,IL-6 and MCP-1 mRNA in the cells were significantly reduced after cantharidin treatment.Compared with the control group,the results of TNF-α and IL-6 in the cell culture medium significantly increased after LPS stimulation(P<0.05),while cantharidin inhibited the above effects of LPS in a concentration-dependent manner(P<0.05).2.Animal experiments: HE staining results showed that compared with the sham operation group,the intimal hyperplasia area and intimal/media ratio of the injured group were significantly increased(P<0.01);Compared with the injury group,the intimal hyperplasia area of the cantharidin group decreased by 62.3%(P<0.01),and the intimal/media ratio decreased by 48.0%(P<0.01).The ELISA results showed that the levels of TNF-α and IL-6 increased in the injured group compared with the sham operation group(P<0.01).Compared with the injury group,the levels of TNF-αand IL-6 in the cantharidin group were significantly decreased(P<0.05).In addition,in the injury group and the cantharidin group,the levels of TNF-α and IL-6 in the 14 days after surgery were lower than those in the 3 days after surgery(P<0.05).The levels of TNF-α and IL-6 in the sham-operated rats did not change significantly after3 days and 14 days after surgery(P>0.05).Immunohistochemical staining showed that the expression of NF-κB p65 in the vascular proliferative intima of the cantharidin group was decreased by 38.4%(P<0.01),the expression of TNF-α and IL-6 decreased by 56.3% and 53.2%,respectively(P<0.05),compared with the injured group.Conclusion:Cantharidin has a significant inhibitory effect on intravascular restenosis,and its mechanism is closely related to its inhibition of NF-κB pathway and reduction of inflammatory response. |
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