| BackgroundAnkylosing Spondylitis(AS)is a chronic inflammatory autoimmune disease that typically occurs in the sacroiliac joints and the attachment points of tendons and ligaments.In the late stage,the fusion of the spine or the peripheral joints may occur,resulting in the loss of the patients' motor function and life ability,and bringing heavy economic burden to the family and society.Inflammation and pathological new bone formation are the two most important pathological features of AS.In the early stage,inflammation and bone erosion and destruction caused by inflammation are the main manifestations,while in the late stage,ectopic new bone formation is caused.Inflammation,as the initiating factor,runs through the whole process of disease development.However,the research on pathological new bone formation and the development of corresponding therapeutic drugs are still in the primary stage.The currently used therapeutic drugs for AS mainly include non-steroidal anti-inflammatory drugs(NSAIDs)and biological agents(TNF-? blockers).Although good anti-inflammatory effects have been achieved,there are still some limitations and side effects,and there is no clear evidence of their effects on the formation of new bone in AS.As first-line drugs recommended by the AS treatment guidelines,NSAIDs have good anti-inflammatory and analgesic effects,but need to be taken for a long time,with side effects such as cardiovascular,gastrointestinal and renal toxicity.Similarly,TNF-? blockers are ineffective for some patients and are expensive,and there are reports that they may increase the risk of developing tumors.Therefore,it is very necessary to develop new drugs for the treatment of AS.Natural products are widely accepted as the main compound in new drug screening studies due to their good chemical diversity,excellent pharmacological activity,relatively low toxicity and low price.As a group of natural products,alkaloids have been widely used in folk Chinese medicine and modern medicine for many years.Sinomenine is one of the alkaloids,which has a wide range of biological activities,and currently known functions include immunosuppression,neuroprotection,memory enhancement,anti-angiogenesis,and anti-tumor.So far,whether sinomenine has a therapeutic effect on AS and its specific mechanism of action has not been reported in the literature.The effect of sinomenine on osteogenic differentiation of cells has not been reported.Therefore,this topic is mainly to explore the effects of sinomenine on AS inflammation and new bone formation and its possible specific molecular mechanisms in vivo and in vitro.ObjectivesPart I: To explore the effects and specific mechanisms of sinomenine on AS inflammation in both cellular and animal models.Part II: To explore the effects of sinomenine on the osteogenic differentiation of bone marrow mesenchymal stem cells.Part III: To explore the specific molecular mechanism of sinomenine inhibiting the osteogenic differentiation of bone marrow mesenchymal stem cells.MethodsPart I:(1)Isolation and culture of monocytes in peripheral blood of patients with AS,stimulating cells with TNF-? to activate inflammatory response,while pre-applying sinomenine to cells,and detecting the expression of cytokines IL-1? and IL-6 in cell supernatant by enzyme-linked immunosorbent assay(ELISA).The activation of NF-?B signaling pathway was detected by western blot.(2)Inducing monocyte differentiation into macrophages,stimulating cells to activate inflammatory response with TNF-?,and pre-applying sinomenine to the cells.The localization of transcription factor p65 in the cells was detected by immunofluorescence(IF).(3)Construct the proteoglycan-induced spinal arthritis model(PGIA),give model mice sinomenine,record each mouse paw swelling rate and mapping,and detect the expression of cytokines IL-1?,IL-6 and IL-17 A in the peripheral blood.Spinal tissue was collected and stained with H&E and Safranin O.The ratio of Th17 cells and Treg cells in the spleen of mice was detected by flow cytometry.Part II:(1)Ficoll density gradient centrifugation was used to isolate and culture bone marrow mesenchymal stem cells from AS patients and normal controls.The purity of the cultured stem cells was identified by observing the cell morphology and proliferation rate,and flow cytometry was used to detect the specific surface markers of stem cells.(2)The osteogenic differentiation of bone marrow mesenchymal stem cells from AS patients and normal controls was induced.The differences in osteogenic ability of stem cells between the two groups were compared by alizarin red S staining and calcium nodules.(3)Osteogenic differentiation of bone marrow mesenchymal stem cells from AS patients was induced.Sinomenine was used to incubate cells.The expression of osteogenic related genes Runx2,ALP and COL1A1 was detected by real-time PCR.ALP staining and activity detection,alizarin red S staining and calcium nodule quantification were used to determine the effect of Sinomenine on osteogenic differentiation of stem cells.Part III:(1)The experiment was divided into negative control group,osteogenic induction group and osteogenic induction + sinomenine group.After 3 days of osteogenic differentiation,transcriptome sequencing was performed,and GO enrichment analysis and Pathway analysis were used to find the biological processes and signaling pathways that sinomenine may be involved in.Then,signal pathways inhibited by sinomenine were further screened through trend analysis.Then,the differential genes in PI3-Akt signaling pathway were analyzed by protein-protein interaction network(PPI)to find the core gene ITGA5.(2)Real-Time PCR and western blot were used to detect the expression of ITGA5 during osteogenic differentiation and sinomenine inhibition of osteogenic differentiation.(3)Drug affinity reaction Target stability(DARTS)was used to detect whether sinomenine directly binds to ITGA5.(4)The expression of ITGA5 was knocked down by siRNA and shRNA,and the effect of ITGA5 on osteogenic differentiation was observed.(5)R language re-analysis of the GEO chip of the spine tissues of the AS mouse model,and explore the expression of ITGA5 in AS.ResultsPart I:(1)Sinomenine inhibited the secretion of IL-1beta and IL-6 cytokines in monocytes induced by TNF-alpha.Western blot results showed that sinomenine inhibits the phosphorylation of transcription factor p65 of NF-kappa B signaling pathway induced by TNF-alpha.ELISA results suggested that sinomenine inhibits the secretion of IL-1beta and IL-6 cytokines in monocytes induced by TNF-alpha.Immunofluorescence results suggested that sinomenine inhibits p65 entry into the nucleus.(2)X-ray of the spine of PGIA mice showed spinal fusion,and H&E staining of the ankle showed destruction and erosion of the joints,which proved the success of the construction of the PGIA model.(3)The paw score curve showed that sinomenine effectively inhibited the inflammation of peripheral joints.H&E and Safranin O staining showed that sinomenine inhibited the erosion of spinal vertebral disc cartilage in PGIA mice.Peripheral blood ELISA showed that sinomenine inhibited the secretion of IL-1?,IL-6 and IL-17 A.Flow cytometry results suggested that sinomenine inhibits the proportion of Th17 cells and increases the proportion of Treg cells.Part II:(1)According to the results of cell morphology and flow cytometry,the extracted bone marrow mesenchymal stem cells were of high purity and could be used for subsequent experiments.(2)The osteogenic differentiation ability of bone marrow mesenchymal stem cells in patients with AS was significantly stronger than that in the normal control group.(3)The optimal concentration of sinomenine(100uM)was screened by CCK8 cell proliferation and toxicity experiments.(4)PCR results demonstrated that sinomenine inhibited the expression of osteogenic related genes Runx2,ALP and COL1A1.ALP staining and activity assays demonstrated that sinomenine inhibited the expression of ALP.Alizarin red S staining and quantitative experiments showed that sinomenine inhibited the formation of calcium nodules.Part III:(1)GO and Pathway analysis of transcriptome sequencing revealed many signaling pathways that sinomenine may be involved in.Further trend analysis revealed that sinomenine may regulate cell osteogenic differentiation by inhibiting PI3K-Akt signaling pathway.The PPI network analysis of the path suggested that ITGA5 is at the core.(2)Western blot and Real-Time PCR results demonstrated that PI3K-Akt signaling pathway and ITGA5 are involved in osteogenic differentiation,and the trends of ITGA5 and PI3K-Akt pathways are consistent.(3)DARTS experiments demonstrated direct binding of sinomenine and ITGA5.(4)After knockdown of ITGA5 by siRNA and shRNA,the process of osteogenic differentiation of bone marrow mesenchymal stem cells was significantly inhibited,indicating that sinomenine inhibited osteogenic differentiation of cells through the ITGA5/PI3K-Akt pathway.(5)Re-analysis of the GEO chip and the immunohistochemical results of the spinal tissues of PGIA mice demonstrated that ITGA5 is highly expressed in the spinal tissue of PGIA model mice.ConclusionsIn this study,three parts of experiments have proved that sinomenine can inhibit inflammation and pathological new bone formation in AS,which provide theoretical support for its simultaneous treatment of AS inflammation and new bone formation.Sinomenine inhibits AS inflammation mainly by regulating NF-kappa B signaling pathway and Th17/Treg balance,and inhibits osteogenic differentiation of bone marrow mesenchymal stem cells by acting on ITGA5/PI3K-Akt pathway.It also suggests that ITGA5 may be a potential therapeutic target for AS pathological new bone formation,which provides strong theoretical support for the development of ITGA5 inhibitors. |
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