Mouse Anti-Human denatured collagen type-I (RGD motif) Monoclonal antibody, clone XL313 Functional Grade

Long non-coding RNAs (lncRNAs) play an important role in the development of bone-related diseases. This study was conducted to investigate the role and mechanism of lncRNA X inactive specific transcript (XIST) in the occurrence of cervical ossification of the posterior longitudinal ligament (OPLL). Here, primary human ligament fibroblasts cells (LFCs) were isolated from 30 cases of OPLL and 30 normal cervical posterior longitudinal ligament (non-OPLL) tissues to perform the qPCR and Western blot assay. We found that the mRNA level of lncRNA XIST was significantly increased in OPLL LFCs compared to non-OPLL LFCs. By bioinformatics analysis, we found that lncRNA XIST has four binding sites for miR-17-5p and found that the mRNA level of miR-17-5p was also significantly decreased in OPLL LFCs compared to non-OPLL LFCs. Since AHNAK is the target gene of miR-17-5p, we further found that the expression of AHNAK was significantly reduced in non-OPLL LFCs after being transfected with miR-17-5p mimic. The qPCR results showed that the mRNA expressions of BMP2 and Runx2 were significantly decreased. After being transfected with lncRNA XIST siRNA in the non-OPLL LFCs, the mRNA levels of lncRNA XIST, AHNAK, BMP2, and Runx2 were significantly decreased and the phosphorylated protein of Smad1/5/8 was reduced. After being cultured by mechanical vibration, the mRNA levels of lncRNA XIST, AHNAK, BMP2, Runx2, COL1, OC, OPN, and Phospho1 were significantly increased, but the mRNA expression of miR-17-5p was significantly decreased. The expression of phosphorylated Smad1/5/8 protein was also significantly increased. Together, this study was the first to determine that XIST gene inhibition plays an important role in the occurrence of cervical OPLL, through the mechanism of regulation of miR-17-5P/AHNAK/BMP2 signaling pathway. Thus, XIST may be a potential target that could be modulated for the treatment of cervical OPLL.

Ethnopharmacological relevance: Forsythiae Fructuse water extract (FSE) is a water-soluble component extracted from the traditional Chinese medicine Forsythiae Fructuse (The fruit of Forsythia suspensa (Thunb.) Vahl) usually used to treat inflammatory diseases. However, little is known about the therapeutic effect of FSE on liver fibrosis. Aim of the study: The purpose of our study was to investigate the therapeutic effect of FSE on liver fibrosis and reveal the underlying mechanism. Materials and methods: Liver fibrosis model was established by subcutaneous injection of olive oil containing 40% CCl4. Rat liver tissue morphologic pathology was investigated by using HE staining, Masson staining and Sirius red staining. Several biochemical markers including liver (ALT, AST, AKP, gamma-GT), fibrosis (HA, LN, PC III, Col IV) and inflammation (IL-6, IL-1 beta, TNF-alpha) were determined by using Elisa kits. Immunohistochemistry was used to observe the distribution of alpha-SMA and COL1 in liver tissue. Effects of FSE on inflammatory pathway (TLR4/MyD88/NF-kappa B) and fibrotic pathway (TGF-beta/smads) were detected by western blot and qPCR. Results: The results showed that hepatic histopathological injury, abnormal liver function, fibrosis and inflammation induced by CCl4 were improved by FSE (2.5, 5 g/kg). Immunohistochemistry and western blot results indicated that the expression of alpha-SMA and COL1 in liver tissue was inhibited by FSE (2.5, 5 g/kg). Western blot and qPCR results further proved that FSE (2.5, 5 g/kg) inhibited the transduction of TLR4/MyD88/NF-kappa B and TGF-beta/smads signaling pathways. Conclusion: FSE can inhibit the expression of inflammatory factors and fibrotic cytokines, reduce liver injury, and inhibit the development of liver fibrosis through TLR4/MyD88/NF-kappa B and TGF-beta/smads signaling pathways.