Effect of naringenin regulating RIP1-RIP3-MLKL signaling pathway on apoptosis of ovarian granulosa cells in rats with polycystic ovary syndrome / 中国药理学通报

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.

Ligand-based substituent-anchoring design of selective receptor-interacting protein kinase 1 necroptosis inhibitors for ulcerative colitis therapy

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 (

RIP1-dependent linear and nonlinear recruitments of caspase-8 and RIP3 respectively to necrosome specify distinct cell death outcomes

There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways. By employing the SWATH-MS technique, we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes. Combining SWATH-MS-based network modeling and experimental validation, we found that when RIP1 level is below ~1000 molecules/cell (mpc), the cell solely undergoes TRADD-dependent apoptosis. When RIP1 is above ~1000 mpc, pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount, which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical observations that RIP1 is required for necroptosis but its increase down-regulates necroptosis. Higher amount of RIP1 (>~46,000 mpc) suppresses apoptosis, leading to necroptosis alone. The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic. Our study provides a resource for encoding the complexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes, enabling RIP1 to play diverse roles in governing cell fate decisions.

The mechanism of anti-hepatocellular carcinoma cell line HepG2 by Chinese medicine Astragalus protein / 药学学报

To detect the inhibitory effect of Astragalus protein on the proliferation of hepatocellular carcinoma cell line HepG2, transcriptomics was used to explore the anti-tumor mechanism of Astragalus protein. The dried roots of Astragalus was precipitated by ammonium sulfate to obtain Huang Qi protein (HQP) with different molecular weights. The effect of HQP on HepG2 and its toxic effect were detected by hemocytometry. Cell necrosis was detected by flow cytometry and Hoechst/propidium iodide (PI) double staining. The necrotic marker protein receptor interacting serine/threonine kinase 1 (RIP1) was determined by Western blot. Transcriptome sequencing was performed on the control group and dosing group RNA, and differential expression genes were analyzed for RNA-seq results. qRT-PCR was used to verified the relative mRNA expression levels of candidate genes. The results showed that the inhibition of HepG2 proliferation was more obvious with the increase of HQP concentration. When the concentration of HQP was 100 μg·mL-1, the necrosis rate increased to 18.78%, and the number of red necrotic cells stained with PI was observed under the microscope. The Western blot results showed an increase in RIP1 protein levels. The results of RNA-seq analysis showed that 26 000 related genes were regulated by HQP, and 979 genes were more regulated. KEGG analysis found that some differentially expressed genes were associated with p53 signaling pathway, and qRT-PCR further verified that the sequencing results were reliable. HQP may cause programmed necrosis of HepG2 cells and may be involved in the p53 signaling pathway.

TNF-α induced RIP1-dependent apoptosis in L929-A cells by interrupting mitochondrial respiratory chain complex Ⅲ / 军事医学

Objective To explore the mechanism by which tumor necrosis factor alpha(TNF-α) induces RIP1 kinase-dependented apoptosis in L929-A fibroblastoma cells.Methods The sub-mitochondrial localization of receptor-interacting protein 1(RIP1),caspase-8 and Bid proteins was detected by dose-gradient trypsin digestion and Western blotting.The levels of reactive oxygen species (ROS),intracellular calcium concentration,mitochondrial membrane potential (MMP),and cellular adenosine triphosphate(ATP) content were determined by fluorescent probe labeling and flow cytometry assay.The mitochondrial respiratory chain complex Ⅰ and Ⅲ activities were detected by commercial kits.Nec-1,A RIP1 kinase specific inhibitor,and RIP1-/-or Bid-/-L929-A cells were used to detect the roles of RIP1 kinase and Bid protein in cell death.Results RIP1,caspase-8 and Bid proteins were co-located in the outer membrane of mitochondrial.TNF-α exposure for 3 h could induce Bid cleavage,inhibit mitochondrial respiratory chain complex Ⅲ activity and reduce MMP.Following these changes and after TNF-α exposure for 6-12 h,the intracellular calcium concentration and ROS were increased,whereas the ATP concentration was decreased,and the cells were killed.Inhibiting RIP1 kinase or knockdown RIP1 or Bid protein could suppress all the cytotoxic effects of TNF-α.Conclusion TNF-α treatment can result in RIP1 kinase-mediated Bid cleavage and inhibit mitochondrial respiratory chains and cell energy metabolism,which ultimately leads to the death of L929-A cells.

Activation of Rip1 promotes necroptosis in LNCaP-AI cells via inhibiting SHARPIN / 中国病理生理杂志

[ ABSTRACT] AIM:To explore the role of SHARPIN in regulation of Rip1 in castration-resistant prostate cancer LNCaP-AI cells.METHODS:The LNCaP-AI cells were treated with TNF-α+Z-VAD ( an inhibitor of pan-caspase) to activate necroptosis, which were compared to the cells treated with TNF-α+Z-VAD+Nec-1 ( an inhibitor of Rip1 ) .A blank group and a TNF-α-treated group were set up as controls.The cell viability in each group was measured by MTS as-say.In addition, SHARPIN was knocked down by siRNA, and the inhibitory efficiency was evaluated by RT-qPCR.The expression of Rip1 at mRNA and protein levels after knocking down SHARPIN was determined by RT-qPCR and Western blot to explore the underlying mechanism of regulatory network of necroptosis in prostate cancer.RESULTS: Compared with blank control group and TNF-α-treated group, the viability of LNCaP-AI cells treated with TNF-α+Z-VAD decreased by 28%(P LNCaP-AI cells.CONCLUSION:Necroptosis is an important way of cell death .Inhibition of oncogenic factor SHARPIN enhances necroptosis via activating Rip1 in LNCaP-AI cells.