基于MALAT1调控VDAC1角度探析丹皮酚改善骨关节炎软骨细胞内质网-线粒体功能紊乱的机制

Mechanism of Paeonol mediates the regulation of VDAC1 by lncRNA MALAT1 to improve endoplasmic reticulum-mitochondrial dysfunction in chondrocytes of osteoarthritis

关键词:  丹皮酚  lncRNA MALAT1  骨关节炎  内质网-线粒体功能

Key words:  Paeonol  lncRNA MALAT1  osteoarthritis  endoplasmic reticulum - mitochondrial function

摘要:

目的 本研究基于长链非编码RNA 转移相关肺腺癌转录本1(lncRNA MALAT1),通过体内和体外实验验证丹皮酚(PAE)调控VDAC1改善骨关节炎(OA)软骨细胞内质网-线粒体功能紊乱的机制。方法 体内实验,C57BL/6小鼠随机分为空白组(10只)和造模组(40只),造模组采用改良Hulth法手术构建OA小鼠模型,随后二次分组为模型组、lncRNA MALAT1过表达组、模型+丹皮酚组、lncRNA MALAT1过表达+丹皮酚组,各组经相关干预后,完成取材。通过HE、番红-固绿、Masson和甲苯胺蓝进行染色观察各组小鼠关节软骨形态学变化。RT-PCR检测软骨组织中lncRNA MALAT1、VDAC1、CHOP、Cyt-C和MMP-13的基因水平表达。Western blot检测关节软骨组织中VDAC1、PERK、CHOP、Bax、Cyt-C、MMP-13蛋白含量表达。体外实验,使用IL-1β诱导软骨细胞构建出符合OA特征的关节炎细胞模型。经慢病毒转染构建lncRNA MALAT1敲减(sh-MALAT1)软骨细胞。通过FISH、RT-PCR和Western blot进一步验证PAE改善IL-1β诱导软骨细胞内质网-线粒体功能紊乱的分子机制。结果 体内实验表明,①PAE可改善OA小鼠关节软骨形态结构。②与模型组相比,模型+丹皮酚组的相关基因和蛋白表达下降(P<0.05),而lncRNA MALAT1过表达可降低PAE的治疗效果。体外实验结果,①FISH显示,PAE可减弱IL-1β干预OA软骨细胞中lncRNA MALAT1的荧光强度。②在IL-1β干预的OA软骨细胞中lncRNA MALAT1、VDAC1、CHOP、Cyt-C和MMP-13基因高表达,PAE干预后,趋势发生逆转。与IL-1β组相比,IL-1β+sh-MALAT1组的相关基因表达下降(P<0.05)。与IL-1β+sh-MALAT1组相比,IL-1β+sh-MALAT1+PAE组的相关基因下降(P<0.05)。③Western blot检测结果显示,PAE可改善IL-1β诱导的sh-MALAT1软骨细胞中相关蛋白的表达(P<0.05)。结论 本研究结果证明PAE可介导lncRNA MALAT1调控VDAC1改善OA软骨细胞内质网-线粒体功能紊乱。

Abstract:

OBJECTIVE This study based on the long non-coding RNA transfer-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1), validated through in vivo and in vitro experiments that Paeonol (PAE) regulates VDAC1 to improve the pathological process of endoplasmic reticulum-mitochondrial dysfunction in osteoarthritis (OA) chondrocytes. METHODS In vivo experiments: C57BL/6 mice were randomly divided into a control group (n=10) and a modeling group (n=40). The modeling group underwent OA modeling via modified Hulth surgery, followed by secondary grouping into model group, lncRNA MALAT1 overexpression group, model+PAE group, and lncRNA MALAT1 overexpression+PAE group. After relevant interventions, samples were collected from each group. The morphological changes of articular cartilage in each group of mice were observed by staining with HE, safranin O-fast green, Masson and toluidine blue. RT-PCR was used to detect the gene expression levels of lncRNA MALAT1, VDAC1, CHOP, Cyt-C and MMP-13 in cartilage tissue. Western blot analysis was performed to detect the expression levels of VDAC1, PERK, CHOP, Bax, Cyt-C and MMP-13 proteins in articular cartilage tissue. In vitro experiments, chondrocytes were induced with IL-1β to establish an arthritis cell model that conforms to the characteristics of OA. lncRNA MALAT1 knockdown (sh-MALAT1) chondrocytes were constructed via lentiviral plasmid transfection. FISH, RT-PCR, and Western blot further validated the molecular mechanism by which PAE ameliorates endoplasmic reticulum-mitochondrial interaction dysfunction in OA. RESULTS In vivo experiments demonstrated: ①PAE can improve the morphological structure of joint cartilage in OA mice. ②Compared with the model group, the model+paeoniflorin group showed reduced expression of relevant genes and proteins (P<0.05), while overexpression of lncRNA MALAT1 diminished the therapeutic effect of PAE. In vitro results: ①FISH shows that PAE can weaken the fluorescence intensity of lncRNA MALAT1 in OA chondrocytes under 1l-1β intervention. ②In IL-1β-treated OA chondrocytes, lncRNA MALAT1, VDAC1, CHOP, Cyt-C and MMP-13 genes were highly expressed; PAE intervention reversed this trend. Compared to the IL-1β group, the IL-1β+sh-MALAT1 group showed reduced expression of these genes (P<0.05). Compared with the IL-1β+sh-MALAT1 group, the IL-1β+sh-MALAT1+PAE group showed reduced expression of the relevant genes (P<0.05). ③Western blot analysis revealed that PAE improved the expression of relevant proteins in IL-1β-induced sh-MALAT1 chondrocytes (P<0.05). CONCLUSION These findings demonstrate that PAE mediates lncRNA MALAT1 regulation of VDAC1 to improve endoplasmic reticulum-mitochondrial dysfunction in OA chondrocytes.