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Aim Based on the apoptotic pathway mediated by receptor interacting protein kinase(RIP)1-RIP3-mixed spectrum kinase domain like protein(MLKL), to explore the effects of naringenin on ovarian granulosa cell apoptosis in rats with polycystic ovary syndrome(PCOS). Methods SD rats were randomly assigned into normal control group, model group, naringenin group, RIP1 inhibitor(Nec-1)group, RIP1-RIP3-MLKL necrosis signal activator(Z-VAD-fmk)group, naringenin+Z-VAD-fmk group, 15 rats per group. ELISA method was performed to measure the levels of IL-1β and TNF-α in ovarian tissue. HE method was performed to observe the shape of the ovary. Granular cells were isolated from ovarian tissue, and flow cytometry was performed to measure apoptosis rate and necrosis rate. Immunohistochemistry was performed to measure the positive expression of p-RIP1 in ovarian tissue. Western blot was employed to detect the expression of RIP1-RIP3-MLKL pathway. Results RIP1 specific inhibitor Nec-1 and naringenin could block the phosphorylation and activation of RIP1, inhibit the RIP1-RIP3-MLKL signaling pathway, reduce the inflammation level in PCOS rats, and alleviate the necrosis and apoptosis of ovarian granulosa cells(P<0.05). Z-VAD-fmk could promote the activation of RIP1-RIP3-MLKL pathway, aggravate the apoptosis of ovarian granulosa cells, and partially weaken the anti-apoptosis effect of naringenin(P<0.05). Conclusions Naringenin may inhibit the apoptosis of ovarian granulosa cells in PCOS rats by blocking the activation of the necrotic apoptotic pathway mediated by RIP1-RIP3-MLKL.
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Osteoarthritis (OA) is the most common chronic disabling joint disease, and currently there is no effective treatment for the cause. Necroptosis plays a key role in many diseases, and receptor-interacting protein kinase 3 (RIP3) is a key regulator during necroptosis process. Studies have shown that the expression level of RIP3 was significantly upregulated in human and mouse OA degenerative cartilage tissues, suggesting the occurrence of necroptosis. However, the specific pathophysiological role of RIP3 in cartilage is still unclear. This study intends to sequence and analyze the transcriptome of chondrocytes before and after RIP3 overexpression, and explore the specific functional mechanism of RIP3 in OA pathogenesis. RNA sequencing results showed that overexpression of RIP3 induced upregulation of 244 genes and downregulation of 277 genes in chondrocytes. Sixteen candidate target genes were screened out by constructing gene co-expression network for further verification at mRNA level, and the results suggested that RIP3 had the most significant inductive effect on the expression of phosphoinositide-3kinase, regulatory subunit 5 (Pik3r5), integrin subunit beta 3 (Itgb3) and MYB proto-oncogene like 2 (Mybl2). Results from CCK-8 and lactate dehydrogenase activity analysis showed that silencing the expression of Itgb3 by siRNA significantly rescued chondrocyte viability decline and necroptosis induced by RIP3, and it also inhibited the upregulating effect of RIP3 on the expression of catabolism-related genes Mmp1, Mmp13 and Il6, as well as the downregulating effect of RIP3 on the expression of anabolism-related genes Acan, Col2a1 and Sox9. This study has demonstrated that RIP3 promotes chondrocyte necrosis and cartilage matrix metabolism disorders by upregulating the expression of Itgb3 in chondrocytes, and ultimately leads to cartilage degeneration. These findings provided potential novel targets for the clinical treatment of OA, and further clarified the pathophysiological significance of necroptosis.
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Receptor-interacting protein (RIP) kinase 1 is involved in immune-mediated inflammatory diseases including ulcerative colitis (UC) by regulating necroptosis and inflammation. Our group previously identified TAK-632 (
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Objective To investigate the effect of baicalin on CT26.WT cells of colon cancer in mice, and to discuss the cell death form. Methods CT26.WT cells were divided into four groups including of control group , routine cultured in fresh medium, the baicalin group, added with concentration of 100 μmol·L-1 baicalin, the z-VAD-fmk group, was added with final concentration of 20 μmol·L-1 z-VAD-fmk, and the combination group, added final concentration of 20 μmol·L-1 z-VADfmk,1 h before adding 100 μmol·L-1 baicalin. Then the inhibitory effect of baicalin on cell proliferation and cell viability were detected by CCK-8 method. The changes of nucleus were detected by DAPI staining, the ultrastructure of cells was observed by TEM, and the effect of baicalin on the expression of RIP3 gene and protein in cells was detected by QPCR method and Western blotting. Results Compared with control group, the differences of baicalin group and combination group had statistically significance (P<0.05) . cell death rate for control group was (10.54±0.19) ％ ,for baicalin group was (34.93±0.16) ％ ,for z- VAD group was (11.23±0.59) ％, and combination group was (23.27±1.20) ％ (P<0.01) . Compared with the normal control group, baicalin group showed nuclear concentration and fragmentation. there was obvious nuclear fragmentation in the combination group against baicalin group. The results of electron microscopy showed that the cells of baicalin were necrotic, cell swelling, mitochondria swelling and contents leaking. Baicalin group significantly up - regulated RIP3 mRNA expression (P < 0. 01) and enhanced RIP3 protein expression (P < 0. 05) . Conclusion Baicalin induces the necrosis of ct26. WT cells, and can significantly increase the gene and protein expression of RIP3.
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Objective:To investigate the expression and function of RIP3 in the liver of rats following ischemic postconditioning.Methods:The model of 70％ hepatic ischemia and reperfusion was established,then a total of forty healthy adult male Sprague-Dawley(SD) rats were divided randomly into four groups,ten rats in each group:a sham-operation group (Sham group);an ischemia reperfusion injury group(IR group);an ischemic postconditioning group(IPO group);an ischemic postconditioning and necrostatin-1 group (Nec-1 group).The blood samples and liver tissues were collected.The serum levels of ALT and AST were detected,and the liver histological examination was performed.Western-bolt was used to detect the TNF-α and RIP3 levels.Results:Compared with the IR group,ALT and AST in serum were significantly declined in the IPO group (P＜0.05);The liver damage after ischemia and reperfusion was improved obviously in the IPO group compared to which in the IR group;The Suzuki's scores was increased in the IR group compared to which in the IPO group (P＜0.05);There was a low grade of TNF-α and RIP3 in the Sham group,whereas the level of TNF-α and RIP3 significantly increased in the IR and IPO and Nec-1 group(P＜0.05);Compared with the IR group,the level of RIP3 was further decreased in the IPO group (P＜0.05);Compared with the IPO group,the level of RIP3 was further decreased in the Nec-1 group (P＜0.05).Conclusion:RIP3-mediated necroptosis was involved during hepatic ischemia postconditioning,and the protective effect of ischemia postconditioning may act as reducing necroptosis by cutting down the levels of RIP3.