| BackgroundKnee Osteoarthritis(OA)is a common and frequently-occurring clinical disease.Its pathological changes mainly include degeneration of cartilage?hyperosteogeny and synovial lesions,leading to joint pain?swelling and other symptoms,which seriously affect the knee function of patients and bring heavy burden to the family and society.At present,there are many clinical treatment methods for OA,but most of them are symptomatic treatment.The discussion of treatment from the level of pathogenesis has always been a hot topic of scholars' attention.Although the exact pathogenesis of OA is still unclear,current studies and most scholars have accepted the view that inflammatory factors are widely involved in the disease process and cause cartilage damage,which is one of the pathogenesis of OA.Pyroptosis is a special programmed cell death mode.Both pyroptosis and OA release a large number of inflammatory cytokines,such as IL-1??IL-18?TNF-? and TGF-?.NLRP3?Caspase-1?IL-18?and IL-1? in pyroptosis associated signaling pathway play an important role.Recent studies have shown that pyroptosis is involved in the occurrence and progression of OA,and inhibition of NLRP3 and Caspase-1 mediated pyroptosis can alleviate OA injury.Therefore,this study focused on the mechanism of bone marrow mesenchymal stem cell(BMSC)derived exosome miR-326 inhibiting pyroptosis of chondrocytes and alleviating cartilage injury,which may provide a new therapeutic idea for clinical treatment of OA cartilage injury.ObjectiveBased on the above research background and previous work,this study intends to study the effect and mechanism of exosome miR-326 derived from bone marrow mesenchymal stem cells(BMSCs)in inhibiting pyroptosis of chondrocytes and alleviating cartilage injury through histone deacetylase(HDAC3),and discuss the possibility of inhibiting pyroptosis of chondrocytes in treating OA.It provides new ideas and approaches for clinical treatment of OA cartilage injury.MethodsIn the following methods,1 is clinical study,2-10 are basic studies,2-9 are chondrocyte level studies and 10 is SD rat level studies.1 Fourteen knee cartilage samples(k-L grade ?)from total knee arthroplasty were collected as the pathological group,and the knee cartilage samples from 5 patients with distal femoral amputation due to trauma were collected as the control group.Meanwhile,peripheral venous blood samples were collected from all patients.To study the pyroptosis of chondrocytes of cartilage in OA patients by hematological index measurement and morphological tissue examination.2 Chondrocytes of knee joint of SD rats were isolated and cultured,and chondrocytes were identified and cultured.3 Bone marrow mesenchymal stem cells(BMSCs)of SD rats were isolated and identified.Exosomes were extracted from BMSCs.The exosomes diameter and morphology were measured,and the expression of marker molecules(CD9?CD63and CD81)were detected.4 BMSCs-Exos and OA rat chondrocytes were co-cultured by WB?PCR?ELISA and immunofluorescence methods.Activity and migration ability of chondrocytes,expression of chondrocytes-specific genes(COL2A1 ? SOX9 ? Agg and Prg4),GSDMD and cleaved caspase-1 in chondrocytes were detected,activity of caspase-1,content of inflammatory cytokines(IL-1??IL-18?IL-6 and TNF-?)and chondrocyte pyroptosis-related proteins(NLRP3?ASC?GSDMD p30?caspase-1p20?IL-1? and IL-18).The objective was to study the effect of BMSCs-Exos on rat chondrocytes.5 Immunofluorescence labeling(PKH26)and PCR were used to determine whether BMSCs-Exos could deliver miR-326 to chondrocytes,and whether BMSCs-Exo could increase the level of miR-326 in chondrocytes of OA rats.6 Mi R-326 mimics and inhibitors were constructed,transfected into BMSC and exosomes were extracted,and then exosomes were co-cultured with OA rat chondrocytes.Using WB?PCR?ELISA and immunofluorescence methods,the activity and migration of chondrocytes,the content of inflammatory cytokines(IL-1??IL-18?IL-6 and TNF-?),the levels of GSDMD and cleaved caspase-1 in chondrocytes,and chondrocyte pyroptosis-related proteins(NLRP3 ? ASC ?GSDMD p30 and caspase-1p20)were detected,miR-326?HDAC3? COL2A1?SOX9? Agg and Prg4 expression levels,NF-?B p65 and caspase-1 activity were detected.HDAC3?STAT1?acetylated STAT1?NF-?B P65 and acetylated NF-?B P65 were detected.The objective was to study the effect of BMSCs-miR-326-Exos on OA chondrocytes.7 On the basis of the above studies,we transfected OA chondrocytes with miR-326 mimics and HDAC3 overexpression vector(HDAC3OE),and BMSCs cells promoted the overexpression of HDAC3 protein on the basis of miR-326,and repeated the indicator detection in Step 6.The objective was to study the effect of HDAC3 overexpression on pyroptosis of OA chondrocytes.8 The specific sites of miR-326 binding to HDAC3 3'-UTR were detected by double luciferase enzyme method.9 OA rat chondrocytes were treated with miR-326 inhibitor and inhibitors of HDAC3 and STAT1 signaling pathway,and the control group was established.The indicator detection in step 6 was repeated.The objective was to study the interaction between miR-326 and Caspase-1 in chondrocytes,and to clarify the effect of inhibitors of HDAC3 and STAT1 signaling pathway on pyroptosis of OA chondrocytes.10 The OA model of rats was established,and the in vivo experiment of OA Rats was conducted to study the effect of BMSCs-miR-326-Exos on pyroptosis of chondrocytes.The histological morphology of cartilage was evaluated by ICRS score? HE and Safranin-O/ Fast Green staining,and the indexes in step 6 were repeated.Results The following results include: 1 is clinical study,2-7 are basic cellular studies,and 8 is basic animal study.1 Clinical studies showed that the levels of Caspase-1and IL-18 in peripheral blood of patients with knee OA in the lesion group were higher than those in the control group,and the differences were statistically significant.Through morphological TUNEL staining,it was found that there was definite chondrocyte pyroptosis in the pathological group,and semi-quantitative statistical analysis showed that the difference between the two groups was statistically significant.2 Effect of BMSCs-Exos on chondrocytes.The activity and migration of chondrocytes ? expression of chondrocyte-specific genes(COL2A1?SOX9?Agg and Prg4)were lower than those of normal chondrocytes.However,caspase-1 activity of chondrocytes in OA rats,the levels of inflammatory cytokines(IL-1??IL-18?IL-6 and TNF-?),the levels of GSDMD and cleaved caspase-1 in chondrocytes,and the chondrocyte pyroptosis-related protein(NLRP3?ASC?GSDMD p30?caspase-1 p20?IL-1? and IL-18)were higher than that of normal chondrocytes.BMSCs-Exos can increase the activity of chondrocyte migration?expression of chondrocyte-specific genes(COL2A1?SOX9?Agg and Prg4).Decreased caspase-1activity in OA rats?the levels of inflammatory cytokines(IL-1??IL-18?IL-6 and TNF-?),and decreased GSDMD and cleaved caspase-1 levels in chondrocytes.Decreased the expression of chondrocyte pyroptosis-related proteins(NLRP3 ?ASC?GSDMD p30?Caspase-1 p20?IL-1? and IL-18).3 Immunofluorescence and PCR showed that BMSCs-Exos delivered miR-326 to chondrocytes,and BMSCs-Exos increased the level of miR-326 in OA chondrocytes.4 Effect of BMSCs-miR-326-Exos on chondrocytes.After BMSCs cells promoted miR-326,their exosomes promoted OA chondrocyte activity and migration ability.Decreased levels of inflammatory cytokines(IL-1??IL-18?IL-6 and TNF-?),GSDMD and cleaved caspase-1 levels,and caspase-1 and NF-?B p65 activity in chondrocytes.Decreased the expression of chondrocyte pyroptosis-related proteins(NLRP3 ? ASC ? GSDMD p30 ?Caspase-1 p20 ? IL-1? and IL-18).Promote the expression of miR-326 and chondrocyte-specific genes(COL2A1 ? SOX9 ? Agg and Prg4)and inhibit the expression of HDAC3.Decreased HDAC3 protein and NF-?B P65 expression,and increased acetylated STAT1?acetylated NF-?B P65 and STAT1 protein expression.BMSCs cells showed opposite results after inhibiting miR-326.5 A chondrocytes were transfected with miR-326 mimics and HDAC3 overexpression vector.Overexpression of miR-326 increased OA chondrocyte activity and migration ability,increased the expression of chondrocyte-specific genes(COL2A1?SOX9?Agg and Prg4),and decreased caspase-1 and NF-?B P65 activities.Decreased the levels of inflammatory cytokines(IL-1??IL-18?IL-6 and TNF-?)? GSDMD and cleaved caspase-1 levels of chondrocytes.Decreased the expression of pyroptosis-related proteins(NLRP3?ASC?GSDMD p30?Caspase-1 p20? IL-1?and IL-18),decreased the expression of HDAC3 protein and NF-?B p65,and increased the expression of acetylated STAT1?acetylated NF-?B P65 and STAT1 protein.Overexpression of HDAC3 protein(HDAC3OE)counteracted the effect of miR-326 mimics,showing opposite results.6 Double luciferase detection results showed that miR-326 binds HDAC3 3'-UTR sites.7 Using HDAC3 and STAT1 signaling pathway inhibitors,the effect of miR-326 on pyroptosis of chondrocytes was studied.The results showed that miR-326 inhibited caspase-1 expression in chondrocytes,and caspase-1 expression was increased by using miR-326 inhibitors.The use of the HDAC3 inhibitor Trichostatin A or STAT1 inhibitor AG490 inhibits the increase of GSDMD and active caspase-1 in miR-326 inhibitor-induced chondrocytes;inhibits the elevation of IL-1??IL-18?IL-6 and TNF-? levels in chondrocytes induced by miR-326 inhibitors;inhibit the mir-326 inhibitor induced elevated levels of NLRP3?ASC?GSDMD-p30?Caspase-1 p20?IL-1? and IL-18 in chondrocytes.8 In vivo test results of OA rats,The ICRS score of cartilage in OA group was significantly lower than that in sham operation group,and the ICRS score of both BMSCs-Exos group and BMSCs-miR-326-Exos group was higher than that in OA group.Both HE and Safranin-O/ Fast Green staining showed that the number?thickness and arrangement of cells in OA group were reduced.The cartilage performance of BMSCs-Exos group and BMSCs-miR-326-Exos group was significantly better than that of OA group.In OA group,the expression of miR-326 and chondrocyte-specific genes(COL2A1?SOX9?Agg and Prg4)decreased,while the expression of HDAC3 increased.The content of cytokines(IL-1??IL-18?IL-6and TNF-?)increased,and the expression of pyroptosis-related proteins(NLRP3?ASC?GSDMD p30?Caspase-1 p20? IL-1? and IL-18)increased.NF-?B p65 and caspase-1 activity increased,GSDMD and Cleaved caspase-1 levels increased,HDAC3 protein and NF-?B p65 expression increased,acetylated STAT1 ?acetylated NF-?B P65 and STAT1 protein expression decreased.The BMSCs-Exos group and BMSCs-miR-326-Exos group reversed this trend in OA group.Conclusion1 Clinical studies have shown that chondrocyte pyroptosis exists in the late stage of human knee OA.2 In vitro cell studies showed that BMSC-Exos could deliver mir-326 to chondrocytes,and BMSCs-miR-326-Exos could inhibit pyroptosis of chondrocytes and maintain chondrocyte homeostasis.Preliminary studies suggest that the mechanism of action is miR-326 targeting the HDAC3 3'-UTR site and inhibiting chondrocyte pyroptosis through the HDAC3 and STAT1/NF-?B p65 pathways.3 In vivo experiments showed that autologous BMSC-derived exosoms miR-326 could inhibit pyroptosis of OA chondrocytes and alleviate OA cartilage injury in rats.4 Upregulation of exosome miR-326,inhibition of chondrocyte pyroptosis and alleviate of cartilage damage through histone deacetylase HDAC3 may be a new idea and measure for clinical treatment of OA. |
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