Studies have demonstrated that FLS highly specialized mesenchymal cells found in the synovium of diarthrodial joints, contribute to the destruction of joint structure in RA. When the joint structure is damaged, the synovial tissue responds as an ill-adapted wound healing response, however if has destructive characteristics and will ultimately lead to irreversible tissue damage to the tendon, cartilage and bone. Therefore, it is important to explore potential therapeutic targets associated with the immune-inflammatory mechanism of the synovial tissue. It has previously been reported that lncRNA MALAT1 is involved in the immune-inflammatory response of RA and is one of the potential therapeutic targets for RA. In this study, we found that the expression level of lncRNA MALAT1 was lower in the CIA group than that in the ctrl group, this was consistent with the downregulation of MALAT1 in peripheral blood mononuclear cells in patients .
In recent years, lncRNA MALAT1 has been one of the star molecules in lncRNA research, with most studies mainly focusing on its tumor immune mechanism. It is considered to have significant lncRNA-targeting potential due to the existence of a highly conserved triple helix structure (ENE) at its 3 'terminal [21, 22]. Therefore, its high conservation is suitable for the study of various immune mechanisms. Studies have been proved that MALAT1 not only regulates apoptosis-related genes by binding to proteins or acting as competitive endogenous RNA, but can also be regulated by drugs to affect apoptosis-related pathways. Herein, results showed that the expression level of MALAT1 was downregulated in RA-FLS in vivo, but its level could be increased by PAE, This finding is consistent with our previous study (currently under review for publication) which found that PAE regulates lncRNA MALAT1 to participate in its treatment of RA in FLS.
In the current study, we explored the therapeutic effect of PAE on CIA rat models. It is worth noting that CIA model has been used extensively to study the mechanism of RA because the model has mature conditions in immunological and pathological features. Our results revealed that PAE reduced the arthritis score. Specifically, histopathology results showed that joint synovial hyperplasia and osteointerstitial inflammatory cell infiltration were reduced after administering PAE, whereas the levels of, the inflammatory cytokines such as TNF –α, IL-6 and IL-2, were all decreased by PAE. Collectively, these findings suggest that PAE has a good therapeutic effect on CIA rat models. A previous study revealed that inflammatory cytokines participate in the pathogenesis of joint inflammatory injury in RA, and there is a wide network of cytokines that contribute to mediating cartilage and bone destruction. TNF –α and IL-6 are both pro-inflammatory cytokines produced by activated RA-FLS. Besides, IL-2, a cytokine glycoprotein, could stimulate T cells and natural killer cells, and take part in the immune response mediated by the Th1 cytokine pathway . It should be noted that the therapeutic effect of PAE increased in a dose-dependent manner. Although several recent studies that have reported that PAE can ameliorate RA joint inflammation by regulating the function of immune cells or related signaling pathways [14, 29, 30], the effect of PAE on MALAT1 has rarely been reported. Based on our previous experimental results, we speculated that PAE may regulate the expression level of MALAT1 to treat RA in FLS. This study found that the arthritis score in the CIA group was higher than that in all PAE treatment groups, which suggests that PAE may reduce the production of inflammatory cytokines and improve synovial tissue inflammation by upregulating MALAT1 in FLS, thereby decreasing joint arthritis scores in CIA rat models.
Considering the unique aggressive phenotype in RA, cell apoptosis plays an important role in the pathological process of RA-FLS . A previous study proved that inducing the apoptosis of RA-FLS may alleviate the development of RA . The TUNEL staining performed in this study displayed that the cell morphology changed, which indicated cell apoptosis, but the number increased after PAE treatment. Moreover, the apoptosis rate was significantly higher in the CIA group with lower expression of MALAT1 than in all PAE treatment groups, suggesting that PAE could improve the apoptosis of RA-FLS by upregulating MALAT1. Furthermore, we explored the expression levels of Bcl-2, Bax, caspase-3 and caspase-9. As one of the conservative cell death pathways, apoptosis is regulated by Bcl-2(B cell Lymphoma 2) family proteins, which composed of pro-apoptotic (such as Bax and Bak) and pro-survival(such as Bcl-2 and Bcl-XL) members . Notably, when the balance between pro-apoptotic and pro-survival factors is broken, caspase 3 and caspase 9 are activatied and mitochondrial cytochromes are released to promote cracking of the cell structure, which ultimately induces cell apoptosis . Herein, qPCR results indicated that PAE treatment could increase the mRNA expression levels of Bax, caspase 3 and caspase 9, and decrease the mRNA expression level of Bcl-2. Meanwhile, western blot analysis results showed that PAE had the same influence on their protein expression level. Overall, these findings suggest that the mechanism through which PAE alleviates the development of RA may associated with the pathway participating in apoptosis.
Accumulating evidence has suggested that the Wnt/β-catenin signaling pathway is activated in the pathogenesis of RA[8, 35]. As a conserved evolutionary pathway, it controls a variety of cellular behaviors in RA. Wnt1, a member of the Wnt ligand gene family, abnormally activates the Wnt/β-catenin pathway and regulates transcription of downstream genes, thereby maintaining cell proliferation, apoptosis, metastasis, and stem cell properties [36–38]. A previous study found that lncRNA MALAT1 affects cell proliferation and apoptosis by inhibiting the Wnt/β-catenin pathway, even including reducing cell inflammatory injury . In this study, the mRNA and protein expression levels of Wnt1 and β-catenin, indicated that the Wnt1/β-catenin pathway was significantly activated in the CIA group, but it was inhibited by PAE in a dose-dependent manner. This result suggested that PAE promotes cell apoptosis in CIA rat models by inhibiting activation of the Wnt1/β-catenin pathway. Moreover, the expression level of MALAT1 was improved in all PAE treatment groups, whereas the Wnt1/β-catenin pathway activity was inhibited in all PAE treatment groups, which further suggests that PAE could enhance inhibition of the Wnt1/β-catenin pathway by upregulating MALAT1.
Results obtained in this study demonstrated that PAE had a beneficial therapeutic effect on arthritis in CIA rats. It was evident that PAE could up-regulate the expression level of lncRNA MALAT1 in FLS of CIA rats, reduce the expression levels of inflammatory cytokines such as TNF –α, IL-6 and IL-2, and inhibit activity of the Wnt1/β-catenin pathway.