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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.
ABSTRACT
Previous studies have shown that high blood glucose-induced chronic microinflammation can cause inflammatory podocyte injury in patients with diabetic kidney disease(DKD). Therein, necroptosis is a new form of podocyte death that is closely associated with renal fibrosis(RF). To explore the effects and mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese herbal medicine Abelmoschus manihot for treating kidney diseases, on podocyte necroptosis and RF in DKD, and to further reveal its scientific connotation with multi-pathway and multi-target, the authors randomly divided all rats into four groups: a namely normal group, a model group, a TFA group and a rapamycin(RAP) group. After the modified DKD rat models were successfully established, four group rats were given double-distilled water, TFA suspension and RAP suspension, respectively by gavage every day. At the end of the 4th week of drug treatment, all rats were sacrificed, and the samples of their urine, blood and kidneys were collected. And then, the various indicators related to podocyte necroptosis and RF in the DKD model rats were observed, detected and analyzed, respectively. The results indicated that, general condition, body weight(BW), serum creatinine(Scr), urinary albumin(UAlb), and kidney hypertrophy index(KHI) in these modified DKD model rats were both improved by TFA and RAP. Indicators of RF, including glomerular histomorphological characteristics, fibronectin(FN) and collagen type Ⅰ(collagen Ⅰ) staining extent in glomeruli, as well as the protein expression levels of FN, collagen Ⅰ, transforming growth factor-β1(TGF-β1) and Smad2/3 in the kidneys were improved respectively by TFA and RAP. Podocyte damage, including foot process form and the protein expression levels of podocin and CD2AP in the kidneys was improved by TFA and RAP. In addition, tumor necrosis factor-α(TNF-α)-mediated podocyte necroptosis in the kidneys, including the morphological characteristics of podocyte necroptosis, the extent and levels of the protein expression of TNF-α and phosphorylated mixed lineage kinase domain like pseudokinase(p-MLKL) was improved respectively by TFA and RAP. Among them, RAP had the better effect on p-MLKL. More importantly, the activation of the receptor interacting serine/threonine protein kinase 1(RIPK1)/RIPK3/MLKL signaling axis in the kidneys, including the expression levels of its key signaling molecules, such as phosphorylated receptor interacting serine/threonine protein kinase 1(p-RIPK1), p-RIPK3, p-MLKL and cysteinyl aspartate specific proteinase-8(caspase-8) was improved respectively by TFA and RAP. Among them, the effect of TFA on p-RIPK1 was superior. On the whole, in this study, the authors demonstrated that TFA alleviates podocyte necroptosis and RF in DKD through inhibiting the activation of the TNF-α-mediated RIPK1/RIPK3/MLKL signaling axis in diabetic kidneys. The authors' findings provide new pharmacological evidence to reveal the scientific connotation of TFA in treating RF in DKD in more depth.
Subject(s)
Humans , Rats , Animals , Diabetic Nephropathies/drug therapy , Abelmoschus , Flavones/pharmacology , Podocytes , Tumor Necrosis Factor-alpha/metabolism , Necroptosis , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Fibrosis , Threonine/pharmacology , Collagen/metabolism , Serine/pharmacology , Diabetes Mellitus/drug therapyABSTRACT
ObjectiveTo observe the effect of Danggui Buxuetang on the podocyte injury and receptor-interacting protein kinase 1/receptor-interacting protein kinase3/mixed lineage kinase domain-like protein （RIPK1/RIPK3/MLKL） signaling pathway in diabetic kidney disease （DKD） ratsand to explore its possible mechanism against DKD. MethodEight of the 50 SD rats were randomly classified intoa normal group， and the remaining were fed a high-glucose and high-fat diet for six weeks and then intraperitoneally injected with 0.035 g·kg-1streptozotocin （STZ） for inducing type 2 diabetes. After successful modeling，they were randomized into the model group，high- and low-dose （1.44，0.72 g·kg-1） Danggui Buxuetang groups， and irbesartan （0.017 g·kg-1）group. After 20 weeks of drug intervention， the fasting blood glucose （FBG）， kidney index （KI），and urinary microalbumin-to-urine creatinine ratio （UACR）were detected in each group. The pathological changes in renal tissue were observed by hematoxylin-eosin （HE） staining， followed by the observation of ultrastructural changes in podocytes under the transmission electron microscope. The levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in renal tissue of rats were determined by enzyme-linked immunosorbent assay （ELISA）， and the protein expression levels of RIPK1， RIPK3， and MLKL in rat kidney tissue by immunohistochemistry. The apoptosis rate of podocytes was detected by in situ nick end-labeling （TUNEL） assay. The mRNA expression levels of RIPK1， RIPK3， and MLKL in kidney tissue of rats were measured by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and the protein expression levels of RIPK， RIPK3， and MLKL and podocyte marker Wilms tumor protein-1 （WT-1） in rat kidney tissue were assayed by Western blot. ResultCompared with the normal group， the model group exhibited elevated FBG， UACR， and KI （P<0.01）， glomerular hypertrophy， thickened basement membrane， increased extracellular matrix， mesangial hyperplasia， foot process fusion or loss， enhanced apoptosis in renal tissue， up-regulated TNF-α and IL-6 levels （P<0.01） and RIPK1/RIPK3/MLKL mRNA and protein expression （P<0.01）， and down-regulated WT-1 protein expression. Compared with the model group， Danggui Buxuetang high-dose group significantly reduced the levels of FBG， UACR， and KI， improved renal histopathology， podocyte loss， and apoptosis in renal tissue， down-regulated TNF-α and IL-6 levels and RIPK1/RIPK3/MLKL mRNA and protein expression （P<0.05， P<0.01）， and up-regulated WT-1 protein expression. ConclusionDanggui Buxuetang alleviates podocyte injury and delays the development of DKD possibly by regulating the RIPK1/RIPK3/MLKL signaling pathway.
ABSTRACT
Necroptosis is a tightly regulated form of necrosis that requires the activation of receptor-interacting protein (RIP) kinases RIPK1 and RIPK3, as well as the RIPK3 substrate mixed lineage kinase domain-like protein (MLKL). Because of membrane rupture, necroptotic cells release damage-associated molecular patterns (DAMPs) that evoke immune responses. Necroptosis is emerging as an important cellular response in the modulation of cancer initiation, progression, and metastasis. Necroptosis of cancer cells is considered to be an immunogenic cell death capable of activating anti-tumor immunity. Necroptosis also participates in the promotion of myeloid cell-induced adaptive immune suppression and thus contributes to oncogenesis. In addition, necroptosis of endothelial cells and tumor cells is conducive to tumor metastasis. In this review, we summarize the current knowledge of the complex role of necroptosis in cancer and discuss the potential of targeting necroptosis components for cancer therapies.
ABSTRACT
Necroptosis is a tightly regulated form of necrosis that requires the activation of receptor-interacting protein (RIP) kinases RIPK1 and RIPK3, as well as the RIPK3 substrate mixed lineage kinase domain-like protein (MLKL). Because of membrane rupture, necroptotic cells release damage-associated molecular patterns (DAMPs) that evoke immune responses. Necroptosis is emerging as an important cellular response in the modulation of cancer initiation, progression, and metastasis. Necroptosis of cancer cells is considered to be an immunogenic cell death capable of activating anti-tumor immunity. Necroptosis also participates in the promotion of myeloid cell-induced adaptive immune suppression and thus contributes to oncogenesis. In addition, necroptosis of endothelial cells and tumor cells is conducive to tumor metastasis. In this review, we summarize the current knowledge of the complex role of necroptosis in cancer and discuss the potential of targeting necroptosis components for cancer therapies.
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Objective:To explore the kinase activity of novel receptor interacting protein kinase 3 (RIPK3) mutants. Methods:The four amino acids (Q84WDF87) of RIPK3 were mutated respectively and these mutants were co-transfected with mixed lineage kinase domain like pseudokinase (MLKL) into HEK293T cells. The auto-phosphorylation of these mutants at S232 and phosphorylation of MLKL at S345 were detected by Western blotting. The interaction between RIPK3 and MLKL was tested by co-immunoprecipitation. The oligomerization of MLKL was detected by non-reducing gel. Results:The kinase activities of RIPK3ΔQ84, RIPK3ΔW85 and RIPK3ΔD86 were effectively decreased. Nevertheless, the kinase activities of RIPK3Q84A/RIPK3Q84E, RIPK3W85Y and RIPK3D86A/RIPK3D86Y did not change markedly. The auto-phosphorylation of RIPK3W85A at S232 was decreased without affecting phosphorylation and oligomerization of MLKL. Conclusion:The amino acid site Q84, W85 or D86 plays a critical role in RIPK3 kinase activity. The kinase activity of RIPK3W85A is decreased, but it does not affect MLKL.
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Objective To identify the role of phosphatidylinositol-3-kinase(PI3K) in mediating necroptosis induced by tumor necrosis factor alpha (TNFα) and the involved mechanism.Methods Knockdown of p110α,receptor-interacting protein 1(RIP1) or both p110αand RIP1 was mediated by the specific short hairpin RNA (shRNA) lentivirus and verified by RT-PCR or Western blotting .In addition , Western blotting was used to detect phosphorylation of mixed lineage kinase domain-like protein(MLKL) and protein kinase B(AKT) or tetramerization of MLKL.Cell death was measured by micros-copy and flow cytometry.Results AKT phosphorylation and TNFα-induced necroptosis of L929 cells were suppressed by the inhibitors of PI3K or AKT, as well as p110αknockdown.Moreover, RIP1 knockdown did not inhibit L929 cell death induced by TNFαplus Z-VAD, but the RIP1-independent necroptosis was inhibited by p 110αknockdown.In addition, p110αknockdown suppressed MLKL phosphorylation and tetramerization induced by TNFαwith Z-VAD in L929 cells. Conclusion PI3K mediates necroptosis of L929 cells induced by TNFαby activating AKT and MLKL, respectively.
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OBJECTIVE To investigate how MLKL functions on the membrane and explore its electrophysiological characters and structure. METHODS The full-length human MLKL were expressed in SF21 cells and purified using glutathione-sepharose affinity chromatography. The currents of purified MLKL proteins were recorded in avoltage-clamp mode using a Warner BC-535 bilayer clamp amplifier. The currents were digitized using pCLAMP 10.2 software. HEK293 cells were cultured and transfected with MLKL plasmid. Cell viability was examined using the CellTiter- Glo Luminescent Cell Viability Assay kit. RESULT MLKL forms cation channels that are permeable preferentially to Mg2+ rather than Ca2+ in the presence of Na+ and K+. Moreover,each MLKL monomer contains five transmembrane helices:H1, H2, H3 , H5 and H6 of the N-terminal domain which is sufficient to form channels. Finally, MLKL-induced membrane depolarization and cell death exhibit a positive correlation to its channel activity.