The Role Of Gsk-3? In Necroptosis

Necroptosis,also known as programmed necrosis,is a form of cell death that is regulated in a Caspase-independent manner and is mainly mediated by RIPK1,RIPK3,and MLKL.Severe tissue damage often bypasses apoptosis and leads directly to necrosis.Inflammatory response can also be strongly induced by necroptotic cell death.Therefore,how to restrain necrosis and reduce inflammation is the focus of the treatment of various tissue injuries.In intestinal inflammation,necroptosis causes cell membrane rupture to release DAMPs(Damage-associated molecular patterns),which contribute to the development of colitis.Glycogen synthase kinase 3(GSK-3),including GSK-3? and GSK-3? two subtypes,was originally found to phosphorylate glycogen synthase and regulate glucose metabolism,as well as play an important role in Wnt signaling.Subsequent studies have revealed that GSK-3 plays an important role in regulating cell differentiation,cell division,apoptosis,and cell proliferation.Although there are numerous studies on necroptosis in recent years,there are still many unknown regulatory mechanisms to be further understood.In the current research,MEF cell lines with GSK-3?inactivation were first treated with the TNF-?/Z-VAD-FMK and cell survival rate was detected by flow cytometry,PI staining and MTT experiment technology.It is found for the first time that GSK-3?inactivation can promote TNF-?/Z-VAD-FMK induced cell death,and this way of cell death can be inhibited by RIPK1 inhibitors Nec-1,so we guess it could be necroptosis.Next,Above cell lines were treated with RIPK1 inhibitor Nec-1 or RIPK3 inhibitor GSK-872 combined with TNF-? /ZVAD-FMK,and the necroptosis markers were detected by Western Blot.The results showed that GSK-3? inactivation would lead to TNF-? /ZVAD-FMK induced activation of necroptosis pathway in MEF cells.Then,the apoptotic markers were detected by Western Blot assay,which ruled out the possibility of apoptosis.Subsequently,mice were modulated by inducing colitis with DSS,and Li Cl was intraperitoneally injected to inhibit GSK-3? activity.The results showed that the inhibition of GSK-3?activity intensified the colon inflammation of mice and increased the mortality of mice.Finally,the activation of necroptosis pathway in the inflammatory intestinal tissues was detected by immunofluorescence and immunohistochemistry assay.