Correlation between the expression of miRNA-142-3p,mixed lineage kinase 3 and interleukin-1beta in nucleus pulposus and the degree of lumbar intervertebral disc degeneration / 中国组织工程研究

BACKGROUND:MicroRNA(miRNA)levels are closely related to cell apoptosis and proliferation,extracellular matrix metabolism and inflammatory response in intervertebral disc cells.However,the specific role of miR-142-3p in lumbar intervertebral disc degeneration remains unclear. OBJECTIVE:To investigate the correlation between the expression of miRNA-142-3p,mixed lineage kinase 3 and interleukin-1β in nucleus pulposus tissue and degree of human lumbar intervertebral disc degeneration. METHODS:A total of 82 patients with lumbar intervertebral disc degenerative diseases in Suzhou Ninth People's Hospital from January 2020 to March 2022 were collected as the study subjects,all of whom underwent MRI examination before operation.According to the Videman classification,the patients were divided into mild degeneration group(n=36),moderate degeneration group(n=26)and severe degeneration group(n=20).Eighty-two specimens of the nucleus pulposus were obtained.The mRNA expression of miRNA-142-3p as well as the mRNA and protein expression of mixed lineage kinase 3,interleukin-1β,type I collagen,type II collagen in nucleus pulposus tissue were detected by qPCR and western blot assay.The correlation between the degree of human lumbar intervertebral disc degeneration and the expression levels of miRNA-142-3p,mixed lineage kinase 3,and interleukin-1β was also assessed using the Spearman correlation coefficient method.Thirty adult Sprague-Dawley rats were divided into sham-operated group(executed after puncturing skin and muscle only),mild degeneration group(executed 1 week after puncturing Co7/8 segments)and severe degeneration group(executed 2 weeks after puncturing Co7/8 segments),with 10 rats in each group.After that,we detected the protein expression of mixed lineage kinase 3 and interleukin-1β as well as the gene expression of miRNA-142-3p,mixed lineage kinase 3 and interleukin-1β in the nucleus pulposus tissue. RESULTS AND CONCLUSION:In human nucleus pulposus tissue,the miRNA-142-3p expression ranked from high to low as follows:mild degeneration group>moderate degeneration group>severe degeneration group(P<0.05);the gene and protein expression of mixed lineage kinase 3 and interleukin-1β from low to high was as follows:mild degeneration group<moderate degeneration group<severe degeneration group(P<0.05);the gene and protein expression of type I collagen from low to high was as follows:mild degeneration group<moderate degeneration group<severe degeneration group(P<0.05),and the gene and protein expression of type I collagen from high to low was as follows:mild degeneration group>moderate degeneration group>severe degeneration group(P<0.05).Spearman correlation analysis showed that the degree of disc degeneration was negatively correlated with miRNA-142-3p expression(P<0.05)and positively correlated with mixed lineage kinase 3 and interleukin-1β expression(P<0.05).In rat nucleus pulposus tissue,compared with the sham-operated group,the expression of mixed lineage kinase 3 and interleukin-1β gene and protein was elevated in the mild degeneration group(P<0.05)while miRNA-142-3p expression was decreased(P<0.05);compared with the mild degeneration group,the expression of mixed lineage kinase 3 and interleukin-1β gene and protein was increased in the severe degeneration group(P<0.05)while miRNA-142-3p expression was decreased(P<0.05).To conclude,the degree of human lumbar intervertebral disc degeneration is negatively correlated with miRNA-142-3p expression and positively correlated with mixed lineage kinase 3 and interleukin-1β expression in nucleus pulposus tissue.

Progress in study on the final executor of necroptosis MLKL and its inhibitors / 中南大学学报(医学版)

Necroptosis is one of the regulated cell death, which involves receptor interacting protein kinase (RIPK) 1/RIPK3/mixed lineage kinase domain like protein (MLKL) signaling pathway. Among them, MLKL is the final execution of necroptosis. The formation of RIPK1/RIPK3/MLKL necrosome induces the phosphorylated MLKL, and the activated MLKL penetrates into the membrane bilayer to form membrane pores, which damages the integrity of the membrane and leads to cell death. In addition to participating in necroptosis, MLKL is also closely related to other cell death, such as NETosis, pyroptosis, and autophagy. Therefore, MLKL is involved in the pathological processes of various diseases related to abnormal cell death pathways (such as cardiovascular diseases, neurodegenerative diseases and cancer), and may be a therapeutic target of multiple diseases. Understanding the role of MLKL in different cell death can lay a foundation for seeking various MLKL-related disease targets, and also guide the development and application of MLKL inhibitors.

Generation of Mlk3 KO mice by CRISPR/Cas9 and its effect on blood pressure / 生物工程学报

To explore the effect of Mlk3 (mixed lineage kinase 3) deficiency on blood pressure, Mlk3 gene knockout (Mlk3KO) mice were generated. Activities of sgRNAs targeted Mlk3 gene were evaluated by T7 endonuclease I (T7E1) assay. CRISPR/Cas9 mRNA and sgRNA were obtained by in vitro transcription, microinjected into zygote, followed by transferring into a foster mother. Genotyping and DNA sequencing confirmed the deletion of Mlk3 gene. Real- time PCR (RT-PCR), Western blotting or immunofluorescence analysis showed that Mlk3KO mice had an undetectable expression of Mlk3 mRNA or Mlk3 protein. Mlk3KO mice exhibited an elevated systolic blood pressure compared with wild-type mice as measured by tail-cuff system. Immunohistochemistry and Western blotting analysis showed that the phosphorylation of MLC (myosin light chain) was significantly increased in aorta isolated from Mlk3KO mice. Together, Mlk3KO mice was successfully generated by CRISPR/Cas9 system. MLK3 functions in maintaining blood pressure homeostasis by regulating MLC phosphorylation. This study provides an animal model for exploring the mechanism by which Mlk3 protects against the development of hypertension and hypertensive cardiovascular remodeling.

Effect of microglia inhibitor Pexidartinib on reconsolidation of remote contextual fear memory in mice / 新乡医学院学报

Objective To investigate the effect of microglia inhibitor Pexidartinib on reconsolidation of remote contextual fear memory in mice.Methods Twelve healthy C57BL/6 male mice were randomly divided into experimental group and control group,with 6 mice in each group.The mice in the two groups underwent contextual fear conditioning training in the contextual fear response box to establish the contextual fear models,and the freezing time of mice after each footshock was recorded.After 7 days,the mice in the experimental group were fed with food containing Pexidartinib formulation PLX3397,while the mice in the control group were fed with regular food until the end of behavioral experiment.On the 16th day after contextual fear conditioning training,the mice were put back into the contextual fear box for recalling the fear memory without any stimulation.The mice were taken out after 5 minutes of free exploration,and the freezing time of mice during this period was recorded.At 24 h after the fear memory was recalled,the mice were again placed in the contextual fear box,allowing them to explore freely for 3 minutes,and the freezing time of mice during this period was recorded;the fear response of mice was indicated by the percentage of freezing time.After the behavioral experiment,the mice were anesthetized by intraperitoneal injection of pentobarbital sodium,and three mice from the two groups were taken and rapidly opened the chest to expose their hearts,the perfusion needle was inserted into the left ventricle from the tip of heart and then was perfuse with 40 g·L-1 paraformaldehyde(pH=7.4)which precooled at 4 ℃ until the involuntary convulsion disappeared and the body limbs of mice were stiff.The brain tissue of mice was taken and fixed with paraformaldehyde solution,and then placed in sucrose solution for dehydration.The brain tissue of mice was coated with tissue embedding agent.The number of microglial cells in the hippocampus of mice in the two groups was detected by immunohistochemistry.The remaining mice in the two groups were taken and quickly decapitated to obtain brain tissues,and the hippocampus tissues of two sides were separated on ice.The expressions of phosphorylated bromodomain-containing protein 4(pBRD4),gasdermin D(GSDMD)and mixed lineage kinase domain-like protein(MLKL)in the hippocampus of mice were detected by Western blot.Results In the stage of contextual fear conditioning training,the percentage of freezing time of mice in two groups increased with the increase of the number of footshock(P＜0.05),but there was no significant difference in the percentage of freezing time of mice after the first,second,third,fourth and fifth footshock between the two groups(P＞0.05).There was no significant difference in the percentage of freezing time of mice between the two groups during recall period(P＞0.05).The percentage of freezing time of mice in the experimental group was significantly lower than that in the control group at the phase of memory test(P＜0.05).The number of microglia in CA1,CA3 and DG regions of hippocampus of mice in the experimental group was significantly lower than that in the control group(P＜0.05).The relative expressions of pBRD4,GSDMD and MLKL in hippocampus of mice in the experimental group were significantly lower than those in the control group(P＜0.05).Conclusion Microglia inhibitor Pexidartinib can injury the reconsolidation of remote contextual fear memory,which may be related to its inhibition of microglial cell activation and the down-regulation of the expressions of pBRD4,GSDMD and MLKL.

Genetic inhibition of MLKL exerts protective effects against cognitive impairment and neuroinflammation in a cerebral small vessel disease model / 中国药理学与毒理学杂志

OBJECTIVE To identify the role of mixed lineage kinase domain like protein(MLKL)in cerebral small vessel disease(CSVD)and explore the underlying mechanism.METHODS Transient bilateral common carotid artery occlusion(tBCCAO)was used to establish a mouse model of CSVD.Immunofluorescence staining and Western blotting were used to observe the expres-sions of RIPK3/MLKL signaling molecules in brain tissues at 7,14 and 28 d after tBCCAO.Open field test,rotarod test,Y-maze and novel object recognition test were used to observe the effect of MLKL knockout on cognitive func-tion after tBCCAO.Blood-brain barrier(BBB)disruption was observed by sodium fluorescein permeability test and the expressions of tight junction proteins.Immunoflu-orescence staining and Western blotting were used to detect the expression of microglia marker Iba-1,astro-cyte marker GFAP,and NLRP3/Caspase-1 signaling mol-ecules in the hippocampus of CSVD mice.ELISA was used to detect the level of inflammatory factors(TNF-α,IL-1β,IL-18)in hippocampus.RESULTS The expres-sions of RIPK3/MLKL signaling molecules increased in cortex and hippocampus after tBCCAO,especially on day 14.The expression of pMLKL mainly increased in neurons,glia cells and endothelial cells in CSVD mice.MLKL knockout improved the cognitive functions such as motor learning,spatial learning and working memory,and object recognition ability in CSVD mice.MLKL knock-out alleviated the leakage of sodium fluorescein and attenuated the down-regulation of tight junction proteins at 1 d and 14 d after tBCCAO.At 14 d after tBCCAO,MLKL knock out inhibited the activations of microglia and astrocytes,attenuated the expressions of NLRP3/cas-pase-1 molecules,and decreased the levels of inflamma-tory factors in the hippocampus of mice.CONCLUSION Genetic inhibition of MLKL exerts protective effects against cognitive impairment by ameliorating BBB dam-age and neuroinflammation in a mouse cerebral small vessel disease model.

Research advances of small molecule inhibitors in the treatment of mixed lineage leukemia / 药学学报

Acute leukemia (AL) is a kind of malignant clonal disease of hematopoietic stem cells. Rearrangement of mixed lineage leukemia (MLL) gene can be observed in about 5%-10% of AL patients. Currently, AL patients with MLL-rearrangements (MLL-r) lack effective treatment and are usually associated with poor prognoses. Recent studies have shown that many epigenetic regulators are directly or indirectly involved in the occurrence and development of AL carrying MLL-r (MLL), which provides a biological basis for the use of epigenetic regulation strategies to treat MLL. In this review, we start from the epigenetic regulation mechanism of MLL, and select representative drug targets to briefly analyze the relationship between each target and MLL and summarize the development progress of their inhibitors, hoping to provide reference for the subsequent research and development of drugs for the treatment of MLL.

Effect of Danggui Buxuetang on Podocyte Injury in Diabetic Kidney Disease Rats： An Exploration Based on RIPK1/RIPK3/MLKL Signaling Pathway / 中国实验方剂学杂志

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.

Effect of effective parts of Dracocephalum moldavica on the necroptosis of HBMECs after OGD/R injury / 药学学报

We investigated the ability of Dracocephalum moldavica (EPDM) flavonoids to protect human brain microvascular endothelial cells (HBMECs) from necroptosis induced by ischemia-reperfusion injury. To mimic the process of cerebral ischemia-reperfusion injury, a necroptosis model was established by treatment with the pan-cysteine aspartic acid protease (caspase) inhibitor Z-VAD-FMK combined with oxygen-glucose deprivation/re-oxygenation (OGD/R) injury using HBMECs. Cell proliferation and cytotoxicity (cell counting kit-8, CCK-8) was used to measure cell viability. A Hoechst33342/PI fluorescent double-staining method was exploited to determine the rate of cell necroptosis. A commercial kit was used to detect lactate dehydrogenase in the cell culture supernate. DCFH-DA probes, calcein AM and JC-1 probes were used to measure changes in ROS production, mitochondrial membrane permeability transformation pore (MPTP) opening and mitochondrial membrane potential (MMP), respectively. Enzyme-linked immunosorbent assay (ELISA) kits were chosen to detect the release of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Western blotting was used to detect necroptosis-related proteins. The results show that relative to control group, Z-VAD-FMK combined with OGD/R injury reduced cell viability, increased the necroptosis rate and the levels of LDH and ROS in HBMECs. The MPTP of the model group cells opened and the MMP reduced. TNF-α, IL-1β, and IL-6 levels were significantly elevated. Furthermore, the expression of receptor-interacting protein kinase 3 (RIP3) and mitochondrial phosphoglycerate mutase 5 (PGAM5) was significantly increased, accompanied by an increase of phosphorylated mixed-lineage kinase domain-like protein (p-MLKL)/MLKL. EPDM partially reversed the changes of the above-mentioned factors in HBMECs induced by Z-VAD-FMK plus OGD/R injury. These results indicate that EPDM may protect HBMECs from cerebral ischemia-reperfusion injury by inhibiting the RIP3/MLKL/PGAM5 pathway and MPTP opening to maintain mitochondrial function, thereby providing a scientific basis for the use of EPDM in the treatment of cerebral ischemia-related diseases.

Programmed Necrosis Inducers for Cancer Treatment / 中国医学科学院学报

Programmed necrosis,a mode of cell death independent of Caspase,is mainly mediated by receptor-interacting protein kinase-1 (RIPK1),receptor-interacting protein kinase-3 (RIPK3),and mixed lineage kinase domain-like protein (MLKL).Studies have demonstrated that programmed necrosis has the dual role of promoting and inhibiting tumor growth and thus we can control the development of tumor by regulating programmed necrosis.The drugs capable of inducing programmed necrosis show potential anti-tumor activity.In addition,inducing programmed necrosis is an effective way to overcome tumor resistance to apoptosis.This paper summarized the mechanisms of programmed necrosis and its relationship with tumors.We focused on the antitumor activity of programmed necrosis inducers including natural products,chemotherapeutic drugs,death receptor ligands,kinase inhibitors,inorganic salts,metal complexes,and metal nanoparticles.These agents will provide new therapeutic candidates for the treatment of tumors,especially the tumors acquiring resistance to apoptosis.

Necroptosis in inflammatory bowel disease: A potential effective target / 中南大学学报(医学版)

The morbidity of inflammatory bowel diseases (IBD) is rising rapidly but no curative therapies to prevent its recurrence. Cell death is crucial to maintaining homeostasis. Necroptosis is a newly identified programmed cell death and its roles played in IBD need to be explored. Necroptosis is mediated by receptor interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL), which resulted in cell swelling, plasma membrane rupture, intracellular content leaking, and eventually cell death as well as the promotion of inflammation. Studies have found that inhibiting necroptosis alleviated IBD in animal models and IBD patients with an increased level of necroptosis in inflammatory tissues, indicating that necroptosis is related to the pathogenesis of IBD. However, due to the complexity in regulation of necroptosis and the involvement of multiple functions of relevant signaling molecules, the specific mechanism remains elusive. Necroptosis may play a vital regulatory role in the pathogenesis of IBD, which provides a new idea and method for further exploring the therapeutic target of IBD.

Effect of programmed necrosis inhibitor Nec-1 on lead-induced BV2 cell injury / 环境与职业医学

Background Programmed necrosis is closely related to the occurrence and development of neurodegenerative diseases, but whether lead causes programmed cell necrosis has not been reported. Objective This experiment is designed to probe into the function of programmed necrosis and the effect of its inhibitor on lead-induced microglia (BV2 cell) injury. Methods The BV2 cells at logarithmic growth phase were treated with 0, 1, 5, 10, 25, 50, 100, and 200 μmol·L−1 lead acetate for 12, 24, 36, and 48 h, respectively, and methylthiazolyldiphenyl-tetrazolium bromide (MTT) was used to determine cell viability. After treatment with 0, 25, 50, and 100 μmol·L−1 lead acetate for 24 h, enzyme-linked immunosorbent assay, Western blotting, and flow cytometry were used to determine the expressions of tumor necrosis factor-α (TNF-α), receptor-interacting protein kinase 3 (RIPK3), receptor-interacting protein kinase 1 (RIPK1), and mixed lineage kinase domain-like protein (MLKL) in the cells, and the effect of RIPK1 inhibitor Nec-1 pretreatment on lead-induced BV2 cell injury . Results The BV2 cell viability decreased with higher lead concentration (r12 h=−0.995, r24 h=−0.984, r36 h=−0.983, r48 h=−0.981, all P<0.01) and time extension (only for 5 μmol·L−1 lead acetate, r=−0.994, P<0.01). Compared with the control group, the BV2 cell viability decreased at the same exposure time when the concentration of lead was above 10 μmol·L−1 (P<0.01). Compared with the control group, the expressions of RIPK1 and MLKL were increased in the 25, 50, and 100 μmol·L−1 lead groups (P<0.05 or 0.01), accompanied by an increase in the contents of inflammatory cytokine TNF-α, especially in the 100 μmol·L−1 lead group, the increment was the highest (P<0.01). The expression levels of p-RIPK1 and p-MLKL in BV2 cells were both increased when the concentration of lead acetate was above 50 μmol·L−1 (P<0.01). In addition, pretreatment with Nec-1 increased the cell viability rate and decreased the necrosis and late apoptosis rate of BV2 cells exposed to lead compared with corresponding lead exposure groups (P<0.05). Conclusions Lead can reduce BV2 cell viability, increase necrosis rate, and up-regulate the expressions of RIPK1, RIPK3, amd MLKL, and the phosphorylation levels of RIPK1 and MLKL. The RIPK1 inhibitor Nec-1 has an intervention effect on lead-induced damage in BV2 cells, indicating that programmed necrosis may play a role in lead neurotoxicity.

Outcome of allogeneic hematopoietic stem-cell transplantation in 50 patients with acutemyeloid leukemia and MLL rearrangement / 中华器官移植杂志

Objective:To investigate the efficacy of allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia associated with 11q23/MLL.Methods:Retrospection and analysis 50 cases of acute myeloid leukemia with 11q23/MLL and who were treated with allogeneic hematopoietic stem cell transplantation(allo-HSCT)in our hospital from September 2012 to December 2019. The efficacy was evaluated by analyzing the transplantation success rate, graft-versus-host disease rate, infection rate, transplant-related mortality(TRM), accumulative recurrence rate, disease-free survival rate(DFS), and overall survival rate(OS).Results:Except for 1 patient had an unsuccessful stem cell transplantationas the result of multiple organ failure, the remaining 49 patients were successfully transplanted. The median time of leukocyte transplantation was 15(9~18)days, and the median time of platelet transplantation was 13(8~33)days. Bone marrow was assessed 28 days after transplantation, and 49 patients were in CR status. The median follow-up time was 38(3~79)months. Between remission group and non-remission group after transplantation, the 3-year OS rates were(83.3±10.8)%, (30.9+ 8.2)%( P=0.002)and the 3-year DFS rates were(83.3+ 10.8)%, (28.4±8.0)%( P=0.003), respectively. Conclusions:Allogeneic hematopoietic stem cell transplantation is an effective method for the treatment of 11q23/MLL rearranged AML. Patients in remission before transplantation have a higher survival rate, and recurrence after transplantation is the primary problem currently faced.

Kinase activity of novel receptor interacting protein kinase 3 mutants / 上海交通大学学报（医学版）

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.

Complex roles of necroptosis in cancer / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

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.

Effect of interferon-γ combined with tumor necrosis factor-related apoptosis-inducing ligand on programmed necrosis of HaCaT cells and its mechanisms / 中华皮肤科杂志

Objective To evaluate the inductive effect of interferon-γ(IFN-γ) combined with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on programmed necrosis of the human immortalized keratinocyte cell line HaCaT,and to explore its mechanisms.Methods In vitro cultured HaCaT cells were divided into several groups:negative control group receiving no treatment,IFN-γ group treated with 50 μg/L IFN-γ,TRAIL group treated with 4 μg/L TRAIL,and cytokine combination group treated with 50 μg/L IFN-γ and 4 μg/L TRAIL or zVAD combination group pretreated with 40 μmo/L zVAD for 1 hour followed by the treatment with 50 μg/L IFN-γand 4 μg/L TRAIL.After 48-hour treatment,the morphology of HaCaT cells were observed under a light microscope,methyl-thiazolyl-tetrazolium assay was performed to evaluate the inhibitory effect of the treatment on the proliferation of HaCaT cells,and double staining flow cytometry to detect the necrosis of HaCaT cells.Meanwhile,real-time fluorescence-based quantitative PCR (qPCR) was conducted to determine the mRNA expression of receptor interaction protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL),Western blot analysis to determine the expression of RIP1,RIP3,MLKL proteins and their phosphorylated forms (pRIP1,pRIP3,pMLKL),immunofluorescent staining to observe the distribution of pRIP3 and pMLKL in HaCaT cells,and the level of reactive oxygen species (ROS) in HaCaT cells in the above groups was detected by the fluorescence probe DCFH-DA.Statistical analysis was carried out with SPSS 22 software by using one-way analysis of variance (ANOVA) for comparing indices among different groups,and least significant difference (LSD)-t test for multiple comparisons.Results After 48-hour treatment,HaCaT cells in the cytokine combination group and zVAD combination group showed necrosis-like morphologic features.Methyl-thiazolyl-tetrazoliumassay revealed significant differences in the survival rate of HaCaT cells among the IFN-γgroup,TRAIL group,cytokine combination group,zVAD combination group and negative control group (73.16％ ± 5.71％,81.46％ ± 4.68％,72.18％ ± 2.93％,69.67％ ± 3.24％ and 100％,respectively;F =24.34,P ＜ 0.001).The necrosis rate of HaCaT cells was notably higher in the cytokine combination group and zVAD combination group (9.86％ ± 1.31％,10.33％ ± 2.16％,respectively) than in the negative control group (5.26％ ± 0.91％,t =4.61,5.07,respectively,both P ＜ 0.05).qPCR revealed that the mRNA expression of RIP3 and MLKL significantly increased in the cytokine combination group and zVAD combination group compared with the negative control group (tRIP3 =0.99,1.84,tMLKL =1.51,2.17,respectively,all P ＜ 0.05).Western blot analysis suggested that the protein expression of RIP1,RIP3,MLKL,pRIP1,pRIP3 and pMLKL significantly increased in the cytokine combination group compared with the negative control group (all P ＜ 0.05),and the zVAD combination group showed significantly decreased caspase 8 expression and increased expression of the above proteins compared with the cytokine combination group.Fluorescence microscopy showed that enhanced green dot-like or clump-like fluorescent spots (representing pRIP3) could be observed in the cytoplasm,and red fluorescent spots (representing pMLKL) could be seen on the cell membrane in the cytokine combination group.The average fluorescence intensity of ROS was significantly higher in the cytokine combination group than in the negative control group (t =702.00,P ＜ 0.05).Conclusion IFN-γcombined with TRAIL can induce the programmed necrosis of HaCaT cells with increased level of ROS.

Complex roles of necroptosis in cancer / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

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.

Prognostic analysis of allogeneic hematopoietic stem-cell transplantation in 47 patients with acute myeloid leukemia and MLL rearrangement / 中华血液学杂志

Objective@#To investigate the prognosis of allogeneic hematopoietic stem-cell transplantation (allo-HSCT) for patients with acute myeloid leukemia and MLL rearrangement.@*Methods@#From September 2009 to May 2016, the clinical data of 47 patients with MLL-rearranged AML undergoing allo-HSCT in the First Affiliated Hospital of Soochow University were retrospectively analyzed.@*Results@#Among 47 MLL-rearranged AML patients, 24 were male and 23 female. The median age was 30 (15-58) years old. There are 36 (76%) patients were FAB-types M4/M5. Two-year overall survival (OS), disease-free survival (DFS), relapse incidence and transplant-related mortality (TRM) were (64.4±8.4)%, (47.3±9.3)%, 41.0% and 17.9%, respectively. Of them, 45 patients were detected with 11q23 translocations, and 2 patients with normal karyotype were MLL partial tandem duplication. According to different chromosome karyotype, 47 patients were divided into three groups: 16 cases of t (6; 11), 15 cases of t (9; 11) and 16 cases of other types. Overall survival was compared between the three groups, there was no significant difference (χ2=1.509, P=0.472). On multivariate analysis, independent risk factor on OS was transplant age >45 years [HR=4.454(95%CI 1.314-15.099), P=0.016]. The multivariate analysis also confirmed the higher TRM in patients at non-CR state when transplanted [HR=10.370(95%CI 1.043-103.110), P=0.046]. Positive minimal residual disease (MRD) before transplantation was a negative prognostic factor on DFS [HR=4.236(95%CI 1.238-14.495), P=0.021] and relapse incidence (RI) [HR=5.491(95%CI 1.371-21.995), P=0.016].@*Conclusion@#Transplant age (>45 years), allo-HSCT in non-CR state adn positive MRD before transplantation were negative prognostic factors in allo-HSCT for MLL-rearranged AML patients.

Expression and regulation of mixed lineage kinase domain-like protein in uterus during early pregnancy and decidua in mice / 第二军医大学学报

Objective To investigate the expression of mixed lineage kinase domain-like protein (MLKL) in uterus during early pregnancy and decidua in mice. Methods Different mouse models including early pregnancy model, artificially induced decidualization model and hormone and/or progesterone treatment of uterine model were constructed; human endometrial stromal cells were cultured in vitro and were induced for decidualization by treating with estradiol-17β, medroxyprogesterone acetate and dibutyryl cyclic adenosine monophosphate. The expression of MLKL mRNA and protein in the uterus of early pregnancy, decidual uterus, and hormone-treated uterus in mice were analyzed by real-time fluorescent quantitative PCR, in situ hybridization and Western blotting. The expression of MLKL mRNA in human decidual cells induced in vitro was detected by real-time fluorescent quantitative PCR. Results (1) In the uterus during early pregnancy in mice, the expression of MLKL mRNA and protein in uterine epithelium on the 1st to 4th day of pregnancy (day 1 was day of vaginal sperm) was low and irregular. It was expressed in the uterine epithelium and surrounding decidual cells on the 5th day of pregnacy, and was mainly expressed in the decidua from the 6th to 8th day of pregnancy. After the implantation, the expression of MLKL mRNA and protein was day-by-day increased and reached the highest on the 7th day of pregnancy, with a slight decrease on the 8th day. (2) In the uterus of mice with artificially induced decidualization, MLKL mRNA was expressed in the entire decidual region with high level; while there was no significant expression in the uterus of the control mice. The expression of MLKL protein was consistent with the expression of MLKL mRNA. The expression of MLKL mRNA in human decidual cells induced in vitro was significantly higher than that in the control group (P0.05). (3) The expression of MLKL mRNA and protein in progesterone-treated uterus was significantly increased compared with the control group (P0.05). Conclusion MLKL regulated by steroid hormone progesterone is involved in embryo implantation and decidualization of mammals.

Prognostic analysis of allogeneic hematopoietic stem-cell transplantation in 47 patients with acute myeloid leukemia and MLL rearrangement / 中华血液学杂志

Objective: To investigate the prognosis of allogeneic hematopoietic stem-cell transplantation (allo-HSCT) for patients with acute myeloid leukemia and MLL rearrangement. Methods: From September 2009 to May 2016, the clinical data of 47 patients with MLL-rearranged AML undergoing allo-HSCT in the First Affiliated Hospital of Soochow University were retrospectively analyzed. Results: Among 47 MLL-rearranged AML patients, 24 were male and 23 female. The median age was 30 (15-58) years old. There are 36 (76%) patients were FAB-types M4/M5. Two-year overall survival (OS), disease-free survival (DFS), relapse incidence and transplant-related mortality (TRM) were (64.4±8.4)%, (47.3±9.3)%, 41.0% and 17.9%, respectively. Of them, 45 patients were detected with 11q23 translocations, and 2 patients with normal karyotype were MLL partial tandem duplication. According to different chromosome karyotype, 47 patients were divided into three groups: 16 cases of t (6; 11), 15 cases of t (9; 11) and 16 cases of other types. Overall survival was compared between the three groups, there was no significant difference (χ(2)=1.509, P=0.472). On multivariate analysis, independent risk factor on OS was transplant age >45 years [HR=4.454(95%CI 1.314-15.099), P=0.016]. The multivariate analysis also confirmed the higher TRM in patients at non-CR state when transplanted [HR=10.370(95%CI 1.043-103.110), P=0.046]. Positive minimal residual disease (MRD) before transplantation was a negative prognostic factor on DFS [HR=4.236(95%CI 1.238-14.495), P=0.021] and relapse incidence (RI) [HR=5.491(95%CI 1.371-21.995), P=0.016]. Conclusion: Transplant age (>45 years), allo-HSCT in non-CR state adn positive MRD before transplantation were negative prognostic factors in allo-HSCT for MLL-rearranged AML patients.

Effects of necroptosis related proteins on brain ischemia/reperfusion injury in mice / 中华行为医学与脑科学杂志

Objective To explore the effect and mechanism of necroptosis related proteins in middle cerebral artery occlusion (MCAO) induced brain ischemia/reperfusion injury in mice.Methods C57BL/6 mice were used to establish the brain ischemia/reperfusion injury model induced by MCAO.MCAO mice were treated with z-VAD.fmk (zVAD,1.1 g/kg),GSK'872 (0.7 g/kg) and combined intervention of zVAD and GSK'872,and neurological defect was evaluated by mNSS while brain infarct volume was measured by TTC staining.Western blot and immunofluorescence assay were used to detect protein expression and location of RIP1,RIP3 and MLKL,respectively.Results Neurological defect and brain infarction were caused by MCAO.Compared with MCAO group,zVAD,GSK'872 and the combined intervention alleviated neurological defect and reduced brain infarct volume significantly (P＜0.05 or P＜0.01).The protein levels of RIP3 and RIP1 MLKL were increased in mice of MCAO group,while GSK'872 and the combined intervention obviously downregulated the aforementioned protein expression [RIP1 (GSK'872:0.64± 0.02 vs MCAO:1.28±0.02,P＜0.01);RIP3 (GSK'872:1.08±0.02 vs MCAO:1.45±0.02,P＜0.01);MLKL (GSK'872:0.54±0.01 vs MCAO:1.00±0.01,P＜0.01)].However,zVAD only slightly reduced protein expression of MLKL (P＜0.05) but didn't change the protein expression of RIP1 and RIP3 (P＞0.05).Conclusion RIP1,RIP3 and MLKL are involved in the execution of necroptosis and contribute to the pathological progress of brain ischemia/reperfusion injury.