CP-25 Improves Vasculitis In HFD CIA Rats By Inhibiting T Cell Function And Endothelial Cell Activation Through Regulating JAKs/STAT3 Signaling Pathway

Rheumatoid arthritis(RA)is a chronic,systemic autoimmune disease.Its main pathological features are arthritis,angiogenesis,synovial hyperplasia,cartilage and bone tissue damage.RA patients have not only joint damage but also other organ damage such as kidney and cardiovascular damage.Cardiovascular diseases(CVDs)are the leading cause of premature death and sudden death in RA.RA is an independent cardiovascular risk factor,and its systemic inflammation is an important factor that ultimately leads to cardiovascular events.Vasculitis caused by endothelial dysfunction plays an important role in CVDs and is a precursor to CVDs events.Endothelial dysfunction is present in both macrovascular and microvascular of RA patients.Endothelial dysfunction is mainly manifested by endothelial cell activation.Activated endothelial cells express adhesion molecules such as intercellular adhesion molecule-1(ICAM-1),and have procoagulant and adhesion-promoting properties.Endothelial cells are one of the main targets of various pro-inflammatory and anti-inflammatory cytokines.Endothelial cell activation is usually induced by pro-inflammatory cytokines such as tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and IL-17,and promotes the recruitment and adhesion of leukocytes.Many studies have focused on the effects of IL-17 on endothelial cell activation.IL-17 is mainly produced by T helper 17(TH17)cells,and the main effector molecule is IL-17A.IL-17A is involved in endothelial cell activation,leading to endothelial dysfunction.Signal transducer and activator of transcription-3(STAT3)is a member of the STAT protein family and an important potential regulator in the inflammatory process.TH17 cells differentiation and regulation are related to STAT3 signaling.More importantly,STAT3 also plays a key role in endothelial cell activation and can increase the expression of ICAM-1.Therefore,understanding the potential relationship between TH17 or IL-17A and the JAKs/STAT3 signaling pathway in the regulation of endothelial cell activation will help to elucidate the molecular mechanism of endothelial cell activation in the RA inflammatory microenvironment.Studies have shown that paeoniflorin(Pae)has vascular protective effects,but the mechanism is unclear.Paeoniflorin-6’-O-benzene sulfonate(CP-25)is an active monomer obtained by structural modification of Pae in our laboratory.Previous studies have shown that CP-25 and Pae exhibit similar anti-inflammatory effects,and the oral absorption of CP-25 is better than that of Pae.CP-25 has immunoregulatory functions,which can improve the histopathological scores of adjuvant arthritis(AIA)rats and collagen-induced arthritis(CIA)mice,inhibit lymphocyte proliferation,and slow down the progression of inflammation.CP-25 regulates the function of B cells through the BAFF-TRAF2-NF-κB signaling pathway.The effect of CP-25 on vasculitis in CIA rats is unknown.Based on the above background,this experiment raises the following scientific questions:Does the JAKs/STAT3 signaling pathway participate in T cells differentiation and endothelial cell activation in CIA rats?Does CP-25 improve vasculitis in CIA rats?Does CP-25 inhibit T cell function and endothelial cell activation through JAKs/STAT3 signaling pathway to improve vasculitis?In this study,CIA rats with high fat diet(HFD)were selected as experimental model animals to observe the therapeutic effect of CP-25 on vasculitis in HFD CIA rats and the effect of CP-25 on the activation and differentiation of T cell subsets.Rat spleen cells were selected in vitro to clarify the molecular mechanism of CP-25 that regulate the JAKs/STAT3 signaling pathway to inhibit T cell function.Human umbilical vein endothelial cells(HUVECs)were selected in vitro as model cells to clarify molecular mechanisms of CP-25 that regulate the JAKs/STAT3 signaling pathway to inhibit endothelial cell activation.Objective:To study the pathogenesis of vasculitis in HFD CIA rats and the therapeutic effect of CP-25 on vasculitis in HFD CIA rats.To clarify the molecular mechanisms that CP-25 improves vasculitis in HFD CIA rats by inhibiting T cell function and endothelial cell activation through JAKs/STAT3 signaling pathway.Methods:1.SD male rats were randomly divided into six groups:Normal group,HFD group,CIA group,HFD CIA group,CP-25 group(50mg/kg),MTX group(0.5mg/kg).After the rats were modeled,the dose was calculated according to the weight of rat.CP-25 group was administered once a day,and MTX was administered twice a week.The duration of administration was 36 days.Evaluate the overall indicators of rats every week,including:global scoring,arthritis index(AI),swollen joint count(SJC);calculate the thymus index and spleen index of rats;CCK-8 method was used to detect the proliferation of spleen B cells and thymic T cells of rats;Stain the spleen,ankle and aorta by HE staining,and observe pathological changes and score.2. Flow cytometry was used to detect the percentage of peripheral blood and spleen T cell subsets in rats at different inflammation stages:CD3~+T cells,CD3~+CD4~+T cells,cells(CD4~+IL-17~+).3. Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels ofIL-17A and ICAM-1 in rat serum,and the biochemical kit was used to detect the levels of serum total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL)and high-density lipoprotein(HDL)of rat.4.Immunofluorescence method was used to detect the expression of p-STAT3 in rat blood vessels.5.Stimulated spleen single cell suspension of rat with cell stimulant in vitro,then add CP-25 to incubate for 24h.Then detect the expression of JAK1,JAK2,JAK3,STAT3,p-JAK1,p-JAK2,p-JAK3,p-STAT3,RORγ-T and IL-17A proteins by western blot(WB).6.HUVECs was stimulated by IL-17A with different concentrations in vitro,detecting the expression of JAK1,JAK2,JAK3,STAT3,p-JAK1,p-JAK2,p-JAK3 and p-STAT3by WB and the secretion of ICAM-1 by ELISA.7.HUVECs was stimulated by IL-17A(50ng/m L)in vitro,and was intervened by CP-25.Then detected the expression of JAK1,JAK2,JAK3,STAT3,p-JAK1,p-JAK2,p-JAK3 and p-STAT3 by WB and the secretion of ICAM-1 by ELISA.Results:1.CP-25 has a therapeutic effect on secondary inflammation and vasculitis in HFD CIA ratsScoring the overall indicators,calculating the spleen index and thymus index,and measuring the proliferation of T cells and B cells,the results showed that compared with the CIA group,the inflammatory response of the HFD CIA rats appeared later,but more severe,and the inflammation subsided slowly.CP-25 can reduce the overall index score,reduce the spleen and thymus index,inhibit the proliferation of T cells and B cells in HFD CIA rats.HE staining results showed that compared with the CIA group,the spleen,ankle,and aortic lesions of HFD CIA rats were severely altered.The aortic endothelium was disordered and locally thickened in HFD CIA rats,and there were local thickening and vacuoles in aorta of HFD CIA rats.CP-25 can significantly improve the spleen,ankle and aortic lesions of HFD CIA rats.2.CP-25 inhibited the differentiation of T cell subsets in HFD CIA ratsThe results of flow cytometry showed that compared with the CIA group,the T-cell subsets in high-fat CIA rats increased slowly in the early stage of inflammation,but the T-cell subsets increased significantly during the peak of inflammation.CP-25can slowly decrease the proportion of T cell subsets in HFD CIA rats,and has certain selectivity for TH17 cells.3. CP-25 inhibited the serum levels of IL-17A and ICAM-1 in HFD CIA rats,and has no effect on blood lipid levels.Serum ELISA results showed that the serum IL-17A and ICAM-1 levels increased most significantly in the HFD CIA group,and CP-25 could significantly reduce the serum IL-17A and ICAM-1 levels in the HFD CIA rats.4. CP-25 inhibited p-STAT3 expression in aorta of ratsImmunofluorescence results showed that the expression of p-STAT3 on the aorta in HFD CIA group most significantly increased,and CP-25 could reduce the expression of p-STAT3 in the aorta to a level close to normal.5. CP-25 inhibited T cell differentiation into TH17 cells by JAKs/STAT3 signaling pathwayCell stimulators were used to stimulate T cells to differentiate into TH17 cells in vitro,and then CP-25 to incubate.The results showed that CP-25 can significantly reduce the expression of p-JAK1,p-JAK2,p-JAK3,p-STAT3,RORγ-T and IL-17A in T cells.6. IL-17A promote endothelial cell activation by JAKs/STAT3 signaling pathwayStimulating HUVECs with IL-17A in vitro,the results showed that IL-17A(50ng/m L)could significantly increase the expression of p-JAK1,p-JAK2,p-JAK3and p-STAT3 and the secretion of ICAM-1.7. CP-25 endothelial cell activation by JAKs/STAT3 signaling pathwayIL-17A was used to stimulate HUVECs in vitro,and then CP-25 was added.The results showed that CP-25 can significantly inhibit the expression of p-JAK1,p-JAK2,p-JAK3 and p-STAT3 and the secretion of ICAM-1.Conclusions:1.JAKs/STAT3 signaling pathway is over-activated in HFD CIA rats,which may promote TH17 cells function and endothelial cell activation.2.CP-25 could inhibit secondary inflammation and vasculitis in HFD CIA rats.3.CP-25 may exert anti-inflammatory effects through inhibiting T cell function and endothelial cell activation.4.CP-25 may inhibit T cell differentiation to TH17 and endothelial cell activation by regulating the JAKs/STAT3 signaling pathway,thereby inhibiting vasculitis.