Effect Of Sox9 On Regeneration Pancreatic β Cells Following Injury

Objective:T1D（type 1 diabetes）is caused by an autoimmune response,in which isletβcells don’t produce enough insulin.Exogenous insulin supplementation can effectively relieve diabetes and its complications,rather than restore damaged isletβcells.SRY-related high mobility group box gene 9（Sox9）is a marker molecule of pancreatic progenitor cells and plays an important role in regulating pancreatic development.The purpose of this study was to investigate the expression characteristics and regulation mechanism of Sox9 in isletβcells induced by repair after injury.Methods:1.After intraperitoneal injection of streptozotocin（STZ）,blood glucose level of SD rats was detected on day 0,2,5,8 and 14;pancreas were collected on 0,8 and 14days after STZ injection for HE staining,and the expression of insulin in islets was detected by immunohistochemical staining;pancreas and islets were collected from control rats and on 14 days after STZ treated rats to detected the expression of Sox9through Western blot,Real time-PCR and immunofluorescent double labelled staining.2.RIN-m5F cells transfected with Lv-EGFP and Lv-r Sox9-EGFP were stimulated by pro-inflammatory cytokines（IL-1βand IFN-γ）for 24 h.Cell morphology was observed under microscope,Western blot was used to detect Bax,Bcl-2,caspase 3 and cleaved-caspase 3 expression.The expression of Bax,Bcl-2 and insulin were detected by Real time-PCR and insulin content in culture supernatant was detected by ELISA.3.Sox9^flox/flox/Ins2-Cre⁺（isletβcells specific Sox9 knockout）mice and Sox9^flox/flox/Ins2-Cre^-mice were injected with STZ to induce isletβcell injury.Fourteen days later,glucose tolerance test was performed,pancreas was collected for HE staining and immunofluorescence staining was used to observe the expression of insulin in islet cells;the expression of apoptosis-related molecules（Bax and Bcl-2）in islet cells was detected by Western blot and Real time-PCR,serum insulin content was detected by ELISA.Results:1.After STZ-induced isletβcells injury in SD rats,the blood glucose levels in rats increased shortly,then gradually returned to near normal levels,the number of isletβcells decreased and then increased,the expression of Sox9 increased in isletβcells when the blood glucose was nearly recovered.2.After 24 h of inflammatory cytokines（IL-1βand IFN-γ）stimulation,compared with RIN-m5F cells transfected with Lv-EGFP,RIN-m5F cells overexpressing Sox9formed fewer cell processes,Bax and cleaved-caspase 3 expression was lower,Bcl-2expression was relatively higher,and insulin secretion levels were also higher.3.Compared to Sox9^flox/flox/Ins2-Cre^-mice,isletβcell-specific knockout Sox9mice(Sox9^flox/flox/Ins2-Cre⁺)decreased glucose tolerance after STZ-induced injury,the number of isletβcells was less and the apoptosis of isletβcells was more obvious,and insulin secretion was severely insufficient.Conclusions:In this study,we further demonstrated that overexpression of Sox9 can inhibit inflammatory factors-induced isletβcells injury and insulin secretion dysfunction based on the discovery of high expression of Sox9 during repair of isletβ-cell injury.Moreover,knockout of Sox9 leads to the failure of timely regeneration induced by injury in isletβcells.These results suggest that Sox9 plays an important role in promoting regeneration of isletβcells induced by injury.