LPS通过上调BMP4促进猪主动脉瓣膜间质细胞成骨样分化

LPS Facilitates Osteogenic Differentiation of Porcine Aortic Valve Interstitial Cells by Upregulating BMP4

该文主要探究了LPS通过上调骨形态发生蛋白4(bone morphogenetic protein 4,BMP4)促进猪主动脉瓣膜间质细胞(valve interstitial cells,VICs)成骨样分化的作用及机制,为钙化性主动脉瓣膜病(calcific aortic valve disease,CAVD)的干预及治疗提供理论依据。采用免疫组化法检测非CAVD组和CAVD组瓣膜组织中Runx2和BMP4的表达,Western blot检测Runx2和BMP4的蛋白表达;采用胶原酶(I型)消化瓣膜后分离猪VICs,用免疫荧光染色进行表型鉴定;采用LPS和重组人BMP4腺病毒处理VICs;用ALP染色、茜素红S染色、qRT-PCR和Western blot法检测细胞的早期和晚期成骨能力、Smad1/5/8和ERK1/2的磷酸化水平。结果显示,BMP4和Runx2蛋白在CAVD组中的表达水平明显高于non-CAVD组。原代猪VICs分离成功,其中α-SMA和Vimentin呈阳性,CD31呈阴性。LPS可使VICs ALP活性增强、钙盐沉积增多、钙化指标上升和BMP4增加;BMP4可使VICs ALP活性增强、钙盐沉积增多、钙化指标上升,且使Smad1/5/8和EKR1/2的磷酸化水平升高。提示LPS可以上调BMP4的表达进而促进VICs的成骨样分化,Smad1/5/8与ERK1/2信号通路可能在该过程中发挥重要作用。

This study aimed to investigate the effect and mechanism of LPS on osteogenic differentiation of porcine aortic VICs(valve interstitial cells)by upregulating BMP4(bone morphogenetic protein 4),which could provide a theoretical basis for the intervention and treatment of CAVD(calcific aortic valve disease).The expression of BMP4 and Runx2 in the non-CAVD group and CAVD group were detected by immunohistochemistry and Western blot.The porcine VICs were isolated by collagenase(type I)after the digestion of the valve,and the phenotype was identified by immunofluorescence staining.VICs were treated with LPS and recombinant human BMP4 adenovirus.ALP(alkaline phosphatase)staining,alizarin red S staining,qRT-PCR,and Western blot were used to detect the early and late osteogenic differentiation abilities of cells.The protein levels of p-Smad1/5/8 and p-ERK1/2 were measured by Western blot.The results showed that the expression of BMP4 and Runx2 in the CAVD group was significantly higher than that in the non-CAVD group.The porcine primary VICs were successfully isolated.The staining ofα-SMA and Vimentin were positive,while the staining of CD31 was negative.LPS significantly increased the ALP activity and the deposition of calcium salts in VICs.The mRNA and protein levels of calcification markers and BMP4 were increased.Besides,BMP4 also increased the ALP activity,the deposition of calcium salts,and the mRNA and protein levels of calcification makers.Meanwhile,the protein levels of p-Smad1/5/8 and p-ERK1/2 were increased by BMP4.In conclusion,LPS promotes osteoblastic differentiation of aortic valve cells by upregulating BMP4.The Smad1/5/8 and ERK1/2 signaling pathways may play important roles in these processes.