1.Effects of Rab7 gene silencing on cytokine and MAPK signal pathway activated by R848 in macrophage
Kai ZOU ; Xiaole YUN ; Hongbin KANG ; Xue WANG ; Xiaohui ZHANG ; Jiming XIE ; Yuzhen WANG
Chinese Journal of Immunology 2017;33(7):967-970
Objective:To investigate the effect of Rab7 on cytokine induced by TLR7 (Toll like receptor-7) R848 activated in Raw264.7,and discusses the influence of Rab7 on MAPK signal transduction.Methods: TLR7 downstream cytokines such as TNF-α,IL-6,IFN-α,IFN-β and IP-10 activated by R848 were detected through Q-PCR in Rab7 silenced mouse macrophages,and then analysis of phosphorylation of MAPK determined with Western blot showed the effect of Rab7 on signal transduction of MAPK.Results: Rab7 inhibit production of cytokine activated by TLR7,and also,Rab7 had an inhibitory effect on MAPK signal pathway.Conclusion: The experimental results further illustrate that the Rab7 is the TLR7 signal transduction pathway negative regulatory factor,and to participate in MAPK signaling pathway.
2.Rab5a promotes LPS-induced cytokine expression in macrophages
Xiaolin SUN ; Jiming XIE ; Xiaole YUN ; Wei ZHANG ; Hongbin KANG ; Yongqing WAN ; Jingran LIU ; Pei GONG ; Shimin ZHAO ; Yuzhen WANG
Chinese Journal of Immunology 2015;(3):295-299
Objective:To establish cell lines stably expressing Rab5a and its the inactive mutant Rab5aN133I,analyze the effect of Rab5a on the expression of cytokines in LPS-stimulated RAW264.7 cells .Methods:RAW264.7 cells were transfected with Rab5a and its inactive mutant vector Rab5a N133I separately,and then screened by G418.Rab5a stable expressing cell lines were identified by Real time-PCR.The growth of the stable cell lines was analyzed by MTT assay.After the stable cell lines were stimulated by LPS for different time periods,the expression of iNOS,TNF-αand IL-6 was detected.Results:Rab5a and Rab5aN133I transfection resulted in elevated Rab5a mRNA expression compared with the control cells ( P<0.05 ).Rab5a overexpression enhanced the proliferation of RAW264.7 cells.However,the proliferation of Rab5aN133I cells was significantly slower than the control cells ( P<0.05).Overexpression of Rab5a promoted LPS-induced production of iNOS,TNF-αand IL-6 in RAW264.7 cells (P<0.01). Conversely,overexpression of Rab5aN133I abolished the stimulating effects of Rab5a.Conclusion: Rab5a promoted LPS-induced expression of iNOS,TNF-αand IL-6 in RAW264.7 macrophages in a GTP-binding ability-dependent manner.
3.Construction and consideration of National Clinical Medical Research Center under the idea of transforming medicine
Henglei DONG ; Xiaoli ZOU ; Huaiyuan XIAO ; Qing LI ; Xiaole XIE ; Hui LI ; Xishan HAO
Chinese Journal of Medical Science Research Management 2018;31(2):81-84
Objective To explore the management of the development of medical research.Methods This paper analyzes the construction mode and experience of the National Center for Clinical Medicine Research in promoting the transformation of medical research,and discusses the cooperation mechanism,talent construction,performance evaluation and institution construction.Results Based on proposed development stage medical institutions and resources,and gradually improve the "transformation chain" management thinking.Conclusions Medical institutions should rely on basic and clin ical resources,based on the concept of medical transformation,strengthen the top design,and gradually change the management model.
4.Mechanism of Cangxitongbi Capsules in Mediating Chondrocyte Pyroptosis in Knee Osteoarthritis via Regulating p38 MAPK/NLRP3/Caspase-1 Pathway
Zhimeng ZHANG ; Daotong YUAN ; Ximin JIN ; Rui GONG ; Zhenlong SONG ; Yongkui ZHANG ; Xiaole WANG ; Rongxiu BI ; Wenpeng XIE
Chinese Journal of Experimental Traditional Medical Formulae 2024;30(22):61-68
ObjectiveTo explore the mechanism of Cangxi Tongbi capsules (CXTB) in regulating the p38 mitogen-activated protein kinase (p38 MAPK)/NOD-like receptor protein 3 (NLRP3)/cysteine protease-1 (Caspase-1) signaling pathway to inhibit pyroptosis of cartilage cells in knee osteoarthritis (KOA). MethodSixty male SD rats were randomly divided into a sham operation group, a model group, low, medium, and high dose CXTB groups, and a positive control group, with 10 rats per group. The modified Hulth method was employed to establish a rat model of KOA. According to their respective assignments, rats were administered CXTB (0.25, 0.5, 1.0 g·kg-1) and Celecoxib (24 mg·kg-1) by gavage. The sham operation and model groups were given an equivalent volume of physiological saline. Treatment was performed once daily for 28 days. Micro-computed tomography (Micro-CT) was used to assess bone volume/total volume (BV/TV) and trabecular separation (Tb.Sp). Joint degeneration was evaluated through hematoxylin-eosin (HE) staining, safranin-fast green (SO) staining, and Osteoarthritis Research Society International (OARSI) scoring. Western blot analysis was conducted to measure the expression levels of p38 MAPK, phosphorylated p38 MAPK (p-p38 MAPK), NLRP3, Caspase-1, and gasdermin D (GSDMD) proteins. Real-time PCR was used to assess mRNA expression levels of p38 MAPK, NLRP3, Caspase-1, and GSDMD genes. Enzyme-linked immunosorbent assay (ELISA) was used to measure serum concentrations of inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-18 (IL-18). After knee replacement surgery, cartilage tissue was analyzed using Western blot to assess the protein expression levels of p38 MAPK, p-p38 MAPK, NLRP3, Caspase-1, and GSDMD, and Real-time PCR was used to evaluate gene expression levels of p38 MAPK, NLRP3, Caspase-1, and GSDMD. ResultMicro-CT analysis revealed significant narrowing of the joint space and increased bone spur formation in KOA rats compared with the sham operation group, with a decrease in BV/TV ratio and an increase in Tb.Sp value (P<0.01). Serum levels of TNF-α, IL-1β, and IL-18 were elevated (P<0.01). The protein expression levels of p-p38 MAPK, NLRP3, Caspase-1, and GSDMD in cartilage were significantly increased (P<0.01), and the mRNA expression levels of p38 MAPK, NLRP3, Caspase-1, and GSDMD were also enhanced (P<0.01). Significant differences in protein expression of p-p38 MAPK, NLRP3, Caspase-1, and GSDMD were observed between normal and diseased cartilage tissues after knee replacement surgery (P<0.05), and the gene expression of p38 MAPK, NLRP3, Caspase-1, and GSDMD were also significantly different (P<0.01). HE and SO staining showed roughened joint surfaces, reduced cartilage thickness, and disordered cellular arrangement in KOA rats. OARSI scores were significantly higher (P<0.01). Compared with the model group, treatment with low, medium, and high concentrations of CXTB resulted in increased BV/TV ratios and decreased Tb.Sp values in the knee joints of rats (P<0.01). HE and SO staining indicated a trend towards smoother joint surfaces and reduced OARSI scores (P<0.01). The protein expression levels of p-p38 MAPK, NLRP3, Caspase-1, and GSDMD were notably decreased (P<0.05), as were the mRNA expression levels of p38 MAPK, NLRP3, Caspase-1, and GSDMD (P<0.01). Additionally, serum concentrations of TNF-α, IL-1β, and IL-18 were significantly reduced (P<0.01). ConclusionCXTB intervention may alleviate knee joint degeneration in KOA rats and inhibit the expression of inflammatory factors and pyroptosis of cartilage cells, thereby protecting cartilage. The underlying mechanism may involve modulation of the p38 MAPK/NLRP3/Caspase-1 signaling pathway.