| Objective: As a cardiopulmonary disease,pulmonary arterial hypertension(PAH)develops rapidly and is difficult to cure.In the course of the disease,pulmonary artery smooth muscle cells(PASMCs)will appear abnormal proliferation,which plays an important role in PAH.The herb Ligusticum chuanxiong has a long history.Ligustrazine extracted from the rhizome of Ligusticum chuanxiong is commonly used to treat cardiovascular diseases,but its effect on PAH is rarely reported.The most commonly used preparation is ligustrazine hydrochloride.Phosphoinositide 3-kinase(PI3K)regulates cell proliferation and it has also been proven to participate in PAH.This study aims to explore whether ligustrazine hydrochloride has therapeutic effect on PAH and whether this effect is related to the regulation of PI3K/AKT pathway.Method: In vivo experiment,a one-time subcutaneous injection of 60mg/kg monocrotaline(MCT)was used to induce PAH in rats,and then ligustrazine hydrochloride(40,80,160mg/kg/day)or sildenafil(25mg/kg/day)was administered for four weeks.(1)Right ventricular systolic pressure(RVSP)was measured using the right ventricular catheterization method.(2)Right ventricular hypertrophy index(RVHI)was measured by weighing method.(3)The morphological characteristics of lung were observed by HE staining.(4)The expression of α-SMA in pulmonary artery was tested by immunohistochemistry.(5)The expression of p-AKT in pulmonary artery was tested by immunofluorescence staining.(6)The expression of proliferating proteins(α-SMA,PCNA),inflammatory cytokine proteins(IL-1β,IL-6,TNF-α),PI3 K and AKT proteins were tested by Western blot.In vitro experiment,primary rat PASMCs were extracted by the tissue adhesion method and identified,a proliferation model was established with platelet-derived growth factor(PDGF)-BB.(1)The effect of ligustrazine hydrochloride on PASMCs proliferation was tested by CCK-8 assay.(2)The effect of ligustrazine hydrochloride on PASMCs cell cycle was tested by flow cytometry.(3)The expression of p-AKT in PASMCs was tested by immunofluorescence staining.(4)The expression of cell cycle associated proteins(PCNA,Cyclin D1,p27Kip1),inflammatory cytokine proteins(IL-1β,IL-6,TNF-α),PI3 K and AKT proteins were tested by Western blot.(5)Besides,after adding the inhibitor(LY294002)to the cells,the effects of them on the PI3K/AKT pathway were tested.Results:(1)the right ventricular catheterization method and organ weighing method,according to the results of ligustrazine hydrochloride(80,160mg/kg)significantly alleviate the RVSP elevation and right ventricular hypertrophy in rats caused by MCT.(2)HE staining results showed that ligustrazine hydrochloride(80,160mg/kg)can obviously improve the pulmonary vascular remodeling in rats caused by MCT.(3)Western blot results showed that ligustrazine hydrochloride(160mg/kg)could significantly improve the proliferation and inflammation of rat pulmonary artery induced by MCT.(4)Immunofluorescence staining and Western blot results showed that ligustrazine hydrochloride(160mg/kg)could reduce the phosphorylation level of PI3K/AKT pathway in rat pulmonary artery induced by MCT.(5)CCK-8 results showed that ligustrazine hydrochloride(10μM)could improve the abnormal proliferation of PASMCs.(6)Flow cytometry results showed that ligustrazine hydrochloride(10μM)could regulate the cell cycle transition of PASMCs.(7)Western blot results showed that ligustrazine hydrochloride(10μM)significantly affected the expression of cell cycle associated proteins and inflammatory cytokine proteins of PASMCs induced by PDGF-BB.(8)Immunofluorescence staining and Western blot results showed that ligustrazine hydrochloride(10μM)could reduce the phosphorylation level of PI3K/AKT pathway in PASMCs induced by PDGF-BB.Conclusion: Therefore,we conclude that ligustrazine hydrochloride may inhibit the proliferation and inflammation of PASMCs by regulating the activation of the PI3K/AKT signaling pathway,thereby attenuating MCT-induced PAH in rats. |
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