Receptor-interacting Protein Serine-threonine Kinases Research Articles

Expression and clinical significance of receptor-interacting protein serine-threonine kinases 1 in the nucleus pulposus of patients with lumbar disc herniation

To investigate the expression and clinical significance of receptor interacting protein serine-threonine kinases 1 (RIPK1) in the nucleus pulposus of patients with lumbar disc herniation (LDH). Nucleus pulposus tissue specimens of 40 patients with LDH patients underwent surgical treatment from January 2016 to January 2018 as the case group, and nucleus pulposus tissue specimens of 30 patients with lumbar spine fracture underwent surgical treatment at the same time as the control group. The expression of RIPK1 mRNA and protein of receptor interaction were detected by polymerase chain reaction (PCR) and Western blot, respectively. The expression of RIPK1 protein in the nucleus pulposus were detected by immunohistochemical staining. The concentrations of RIPK1 and tumor necrosis factor-α (TNF-α) in nucleus pulposus were detected by ELISA method. The relationship between the concentrations of RIPK1, TNF-α in nucleus pulposus and the Pearce grade of LDH patients was analyzed by one-way ANOVA. The correlation between RIPK1 and TNF-α was analyzed by Pearson. RIPK1 was weakly positively expressed in nucleus pulposus of control group, and RIPK1 protein was positively or strongly positively expressed in case group. The expression of RIPK1 mRNA in nucleus pulposus of case group was higher than that of control group (P<0.05). The expression of RIPK1 protein in nucleus pulposus of case group was higher than that of control group(P<0.05). The RIPK1 concentration in nucleus pulposus of case group was higher than that of control group (P=0.012). The concentration of TNF-α in nucleus pulposus of case group was significantly higher than that of control group (P=0.009). There were significantdifferences in concentrations of RIPK1 and TNF-α in nucleus pulposus of LDH patients with different Pearce classification (P>0.05). The concentration of RIPK1 and TNF-α in nucleus pulposus increased significantly with the increase of Pearce grade. Pearson correlation analysis showed that there was a significant positive correlation between RIPK1 and TNF-α in nucleus pulposus of LDH patients (r=0.781, P<0.001). The expression levels of RIPK1 mRNA and protein in the intervertebral disc tissues of LDH patients are higher than those of normal intervertebral disc tissues, and increased with the increase of Pearce grade, which may be an important factor involved in LDH inflammatory disease.

Extracts derived from a traditional Chinese herbal formula triggers necroptosis in ectocervical Ect1/E6E7 cells through activation of RIP1 kinase

Ethnopharmacological relevanceAs one of the most common female malignant tumors mainly infected by human papillomavirus (HPV) worldwide, cervical cancer is widely distributed in about 90% developing countries. An in-hospital preparation derived from a traditional Chinese herbal formula, Youdujing (YDJ), has been developed and clinically used for more than 20 years in our hospital for treating multiple diseases caused by HPV infection, such as cervical precancerous lesions, recurrent condyloma acuminata, fla t warts, etc. However, few investigations on the effect and mechanism of YDJ extract on treating and preventing HPV infection induced cervical cancer have been reported. Aim of the studyPrevious reports showed that YDJ extract is effective in triggering human cervical cancer cells (ectocervical Ect1/E6E7) death in a necrotic manner. Herein, we aim to investigate the anti-proliferation effects and potential mechanisms of YDJ extract in inducing necroptosis in ectocervical Ect1/E6E7 cells. Materials and methodsThe high-performance liquid chromatography (HPLC) fingerprint method was firstly used for better quality control of the chemical components in YDJ extract. MTT assay and flow cytometer were applied for evaluating cytotoxicity and necroptosis induced by YDJ extract in ectocervical Ect1/E6E7 cells. Besides, Western blotting, receptor-interacting protein serine-threonine kinase 1 (RIP1) inhibitor (necrostatin-1), and RIP1 shRNA and pCDNA transfection assays were employed for investigation on the underlying mechanisms and validation the role of RIP1 in YDJ extract induced necroptosis. ResultsYDJ extract induced necroptosis in ectocervical Ect1/E6E7 cells both in time- and concentration-dependent manners, without affecting activation of caspases and elevation of intracellular reactive oxygen species (ROS) level. Moreover, a selective increasing in RIP1expression was observed in YDJ extract treated ectocervical Ect1/E6E7 cells. The induction effect of necroptosis by YDJ extract was partially blocked by the addition of RIP1 inhibitor (necrostatin-1). Co-immunoprecipitation assay demonstrated that the treatment of YDJ extract in ectocervical Ect1/E6E7 cells promoted the combination of RIP1 with RIP3 and MLKL to form necrosome, which facilitates the process of necroptosis. ConclusionsTaken together, YDJ preparation displays an effective ability of inducing necroptosis in cervical cancer cells through activation of RIP1 kinase. However, although the treatment efficacy and potential mechanisms of YDJ extract in vivo remain unclear and need further investigation, it is believed that YDJ extract has the great potential to be used as a starting point to develop more potent agent for treating or preventing cervical cancer and other proliferative diseases.

Relationship between mechanical ventilation-induced apoptosis in hippocampal neurons and mTOR signaling pathway in mice

Objective To evaluate the relationship between mechanical ventilation-induced apoptosis in hippocampal neurons and mammalian taget of rapamycin(mTOR)signaling pathway in mice. Methods Fifty healthy male C57BL/6 mice, aged 8-10 weeks, weighing 20-25 g, were divided into 2 groups (n=25 each) using a random number table method: control group (group C) and mechanical ventilation group (group V). The mice breathed spontaneously for 6 h in group C, and the mice were mechanically ventilated for 6 h in group V. Open field test and contextual fear conditioning test were conducted at 1 and 3 days after the end of ventilation.Hippocampal tissues were obtained at 1 day after the end of ventilation for determination of the expression of mTOR, phosphorylated mTOR (p-mTOR), microtubule-associated protein 1 light chain 3 Ⅱ(by Western blot)and apoptosis in hippocampal neurons (by TUNEL). The p-mTOR/mTOR ratio and apoptosis index were calculated. Results Compared with group C, the time animals spent in the central square was significantly prolonged, the number of crossing the grid was reduced, the percentage of freezing time was decreased, the expression of microtubule-associated protein 1 light chain 3 Ⅱ was up-regulated, and the p-mTOR/mTOR ratio and apoptosis index were increased in group V (P<0.05). Conclusion The mechanism by which mechanical ventilation induces apoptosis in hippocampal neurons may be related to activation of mTOR signaling pathway in mice. Key words: Respiration, artificial; Apoptosis; Receptor-interacting protein serine-threonine kinases; Cognition disorders

Loss of periodontal ligament fibroblasts by RIPK3-MLKL-mediated necroptosis in the progress of chronic periodontitis

Periodontal homeostasis is maintained by the dynamic equilibrium between cell death, differentiation and proliferation of resident cells in the periodontal microenvironment. Loss of resident periodontal ligament fibroblasts (PDLFs) has been a major challenge in the periodontal treatment. This study aimed to investigate the exact role of necroptotic cell death in periodontal diseases. Elevated levels of receptor-interacting protein serine-threonine kinases -1 (RIPK1), phosphorylated RIPK3, mixed lineage kinase domain-like protein (MLKL), phosphorylated MLKL and FLIPL were observed in gingival tissues collected from patients with untreated chronic periodontitis; whereas no difference in caspase 8 was observed between the periodontitis and healthy control group. In contrast to the high incidence of necroptotic cell death in monocytes during live P. gingivalis infection with a low multiplicity of infection (MOI), necroptosis was only observed in PDLFs with a high MOI. Priming PDLFs with frozen thawed monocytes enhanced proinflammatory responses to P. gingivalis infection; moreover, frozen thawed monocytes stimulation triggered RIPK1, RIPK3 and MLKL-mediated-necroptotic cell death in PDLFs. These results indicated that RIPK3 and MLKL-mediated-necroptotic cell death participated in the pathogenesis of periodontitis, and DAMPs released from monocytes after P. gingivalis stimulation by necroptosis triggered not only inflammatory responses, but also necroptosis of PDLFs.

Role of mammalian target of rapamycin in synaptic plasticity of entorhinal area-hippocampal formation in rats with inflammatory pain

Objective To evaluate the role of mammalian target of rapamycin(mTOR) in the synaptic plasticity of entorhinal area-hippocampal formation in rats with inflammatory pain. Methods Twenty-four healthy adult male Sprague-Dawley rats, weighing 180-240 g, were divided into 4 groups(n=6 each) by using a random number table method: control group(group C), inflammatory pain group(group IP), dimethyl sulfoxide(DMSO) group and mTOR inhibitor rapamycin group(group R). Inflammatory pain model was established by subcutaneous injection of 50 μl bee venom into the plantar surface of the left hindpaw. The equal volume of normal saline was subcutaneously injected into the plantar surface of the left hindpaw in group C. In group DMSO, 2% DMSO was administered by intragastric gavage for 3 days, 1 ml per day, and the inflammatory pain model was established at 1 h after administration on 3rd day. In group R, rapamycin was administered by intragastric gavage for 3 days, 1 ml per day, and the inflammatory pain model was established at 1 h after administration on 3rd day. Mechanical paw withdrawal threshold(MWT) and thermal paw withdrawal latency(TWL) were measured at 2 h after establishing the model. The rats were sacrificed after measurement of the pain threshold, and hippocampi were removed to prepare hippocampal slices. Hippocampal CA1 region and dentate gyrus(DG region) were located with an inverted microscope. Planar microelectrode array technique was used to record the number of channels and the standardized amplitude of evoked effective field excitatory postsynaptic potentials(fEPSPs)(fEPSPs amplitude>20% of the baseline value) at different stimulus intensities. Results Compared with group C, MWT was significantly decreased, TWL was shortened, the number of effective fEPSP channels at different stimulus intensities was increased, and the amplitude of standardized fEPSPs in hippocampal DG and CA1 regions was increased in group IP(P 0.05). Compared with group IP, MWT was significantly increased, TWL was prolonged, the number of effective fEPSP channels at different stimulus intensities was decreased, and the amplitude of standardized fEPSPs in hippocampal DG and CA1 regions was decreased in group R(P 0.05). Conclusion mTOR is involved in the changes in the synaptic plasticity of entorhinal area-hippocampal formation in rats with inflammatory pain. Key words: Receptor-interacting protein serine-threonine kinases; Hippocampus; Entorhinal cortex; Pain; Inflammation; Neuronal plasticity

Role of class I histone deacetylase in myocardial ischemia-reperfusion injury in diabetic rats and the relationship with AMPK/mTOR signaling pathway

Objective To evaluate the role of class Ⅰ histone deacetylase (HDAC) in myocardial ischemia-reperfusion (I/R) injury in diabetic rats and the relationship with adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway. Methods SPF healthy adult male Sprague-Dawley rats, weighing 210-220 g, were used in this study.Type I diabetes mellitus was induced by single intraperitoneal injection of streptozotocin dissolved in citrate buffer 60 mg/kg, and 8 weeks later the rats with type I diabetes mellitus were used for experiment.Forty-eight diabetic rats were divided into 4 groups (n=12 each) by using a random number table method: sham operation group (group S), myocardial I/R group (group I/R), myocardial I/R plus class I HDAC inhibitor MS-275 group (group I/R+ MS) and myocardial I/R plus MS-275 plus AMPK inhibitor Compound C group (group I/R+ MS+ CC). Myocardial I/R was induced by ligation of the left anterior descending branch of the coronary artery for 45 min followed by 180 min of reperfusion in anesthetized rats.In group I/R+ MS, MS-275 10 mg/kg was intraperitoneally injected once a day for 7 consecutive days, and myocardial I/R was produced after the end of administration.AMPK inhibitor Compound C 0.5 mg/kg was intravenously injected at 30 min before ischemia in group I/R+ MS+ CC.Six rats were sacrificed at the end of reperfusion for determination of myocardial infarct size.Another 6 rats were selected at the end of reperfusion and sacrificed for determination of the level of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) in serum (by enzyme-linked immunosorbent assay), expression of AMPK, phosphorylated AMPK (p-AMPK), mTOR, phosphorylated mTOR (p-mTOR), ubiquitin-binding protein P62 (P62), microtubule-associated protein 1 light chain 3 Ⅰ (LC3 Ⅰ) and LC3Ⅱ in myocardial tissues (by Western blot). The ratios of p-AMPK/AMPK, p-mTOR/mTOR and LC3Ⅱ/Ⅰ were calculated. Results Compared with group S, the myocardial infarct size and levels of serum CK-MB and LDH were significantly increased in I/R, I/R+ MS and I/R+ M+ CC groups, the ratios of p-AMPK/AMPK and LC3Ⅱ/Ⅰ were significantly increased, p-mTOR/mTOR ratio was decreased, and P62 expression was down-regulated in group I/R+ MS (P 0.05). Compared with group I/R, the myocardial infarct size and levels of serum CK-MB and LDH were significantly decreased, the ratios of p-AMPK/AMPK and LC3Ⅱ/Ⅰ were increased, p-mTOR/mTOR ratio was decreased, and P62 expression was down-regulated in group I/R+ MS (P 0.05). Compared with group I/R+ MS, the myocardial infarct size and levels of serum CK-MB and LDH were significantly increased, the ratios of p-AMPK/AMPK and LC3Ⅱ/Ⅰ were decreased, p-mTOR/mTOR ratio was increased, and P62 expression was up-regulated in group I/R+ M+ CC (P<0.05). Conclusion Class Ⅰ HDAC is involved in myocardial I/R injury through enhancing AMPK/mTOR signaling pathway-regulated level of autophagy in diabetic rats. Key words: Histone deacetylase; Autophagy; Diabetes mellitus; Myocardial reperfusion injury; Protein-serine-threonine kinases; Receptor-interacting protein serine-threonine kinases

Effect of bone marrow-derived mesenchymal stem cells on mTOR signaling pathways in lung tissues of rats with acute lung injury

Objective To evaluate the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on mammalian target of rapamycin (mTOR) signaling pathways in lung tissues of rats with acute lung injury (ALI). Methods Healthy pathogen-free adult male Sprague-Dawley rats were selected, and the BMSCs were obtained and cultured in vitro.One hundred and five healthy clean adult male Sprague-Dawley rats, weighing 170-190 g, were divided into 5 groups (n=21 each) using a random number table: control group (group C), PBS group, group ALI, ALI plus BMSC group (group ALI+ BMSCs), and ALI plus phosphate buffer solution (PBS) group (group ALI+ PBS). Group C received no treatment.PBS 0.5 ml was injected via the tail vein in group PBS.Lipopolysaccharide (LPS, 0.5 ml) 5 mg/kg was intraperitoneally injected to establish the model of ALI in group ALI.BMSCs (0.5 ml) 1×104 cells/ml were injected via the tail vein after intraperitoneal injection of LPS in group ALI+ BMSCs.PBS 0.5 ml was injected via the tail vein after intraperitoneal injection of LPS in group ALI+ PBS.Arterial blood samples were collected for blood gas analysis at 6, 24 and 48 h after injection of BMSCs.Lungs were then removed for determination of wet/dry weight ratio (W/D ratio) and expression of mTOR, nuclear factor kappa B (NF-κB) and tumor necrosis factor-alpha (TNF-α) in lung tissues (by Western blot) and for examination of the pathologic changes of lungs tissues (using haematoxylin and eosin staining). Results Compared with group C, pH value and PaO2 were significantly decreased, PaCO2 and W/D ratio were increased, and the expression of mTOR, NF-κB and TNF-α was up-regulated at each time point in ALI, ALI+ BMSCs and ALI+ PBS groups (P<0.05). Compared with group ALI, pH value and PaO2 were significantly increased, PaCO2 and W/D ratio were decreased, the expression of mTOR, NF-κB and TNF-α was down-regulated at each time point (P<0.05), and the pathologic changes of lungs tissues were significantly attenuated in group ALI+ BMSCs. Conclusion The mechanism by which BMSCs reduce ALI may be associated with inhibiting mTOR signaling pathways in lung tissues of rats. Key words: Mesenchymal stem cell transplantation; Receptor-interacting protein serine-threonine kinases; Respiratory distress syndrome, adult

Receptor interacting protein serine threonine kinase 3 mediates macrophage activation and interleukin-6 production in autoimmune hepatitis

Objective To investigate the expression level of receptor-interacting protein serine threonine kinase (RIP) 3 in macrophages/monocytes activation in autoimmune hepatitis (AIH) and its regulation on inflammatory cytokines. Methods The degree of macrophage infiltration and the expression of RIP3 in liver tissues from patients with AIH or hepatic cysts by double-immunofluorescence. After 24 hours treated with different concentrations (0, 1, 3, 6, 10 μg/mL) of lipopolysaccharide (LPS), Necrostatin-1, the specific inhibitor of RIP3 signaling pathway, and 6-thioguanine, the active metabolite of azathioprine, the expression levels of RIP1 (RIP3 upstream signal molecule), RIP3 and mixed-lineage kinase domain-like protein (MLKL, RIP3 downstream substrate) in RAW264.7 macrophages were detected by Western blotting. The expression of macrophage-associated cytokine at mRNA level of each treatment group was determined by real-time quantitative polymerase chain reaction (PCR). Student′s t test and sum rank test were performed for statistical analysis. Spearman analysis was performed for the correlation analysis. Results Compared with hepatic cysts adjacent liver tissues, the infiltration of CD68 positive macrophages in liver tissue of AIH patients was significantly increased (4.75±0.96 vs 28.86±6.23), and the difference was statistically significant (t=7.80, P<0.05), and the expression level of RIP3 also was significantly increased (15, 11 to 22 vs 0, 0 to 1), and the difference was statistically significant (Z=-2.66, P<0.05). In vitro, compared with those of control group, the expression levels of RIP1, RIP3 and MLKL stimulated by LPS at 0, 1, 3, 6, 10 μg/mL were significantly increased, and the differences were statistically significant (t=4.00, 4.90, 6.40, 10.30; 3.80, 9.30, 9.80, 9.00; 4.90, 9.90, 9.30 and 7.70; all P<0.05), and were dose-dependent (r=0.91, 0.86 and 0.79, all P<0.05). Furthermore, compared with those of LPS-stimulated group, the expressions of RIP1, RIP3, MLKL of LPS+ Necrostatin-1 group were significantly decreased (0.73±0.11 vs 0.47±0.13, 0.60±0.07 vs 0.37±0.05, 0.65±0.22 vs 0.38±0.04, respectively), and the differences were statistically significant (t=2.60, 4.50 and 2.10, all P<0.05). And the expressions of interleukin (IL)-1β, IL-6 and IL-10 at mRNA levels were also decreased (810.3±200.8 vs 463.7±118.1, 1 504.4±482.7 vs 290.4±106.9, 1 358.6±559.2 vs 677.8±297.6, respectively), and the differences were statistically significant (t=5.40, 12.52, 5.70, all P<0.05). However, the expressions of IL-4 and TGF-β at mRNA levels up-regulated (0.3±0.2 vs 0.6±0.3, 0.4±0.1 vs 0.9±0.4, respectively), and the differences were statistically significant (t=4.60 and 6.10, both P<0.05). Compared with those of the LPS-stimulated group, the expressions of IL-1β, IL-6 and IL-10 at mRNA levels of LPS and 6-thiopurine stimulated group significantly down-regulated (810.3±200.8 vs 283.4±65.5, 1 504.4±482.7 vs 354.4±73.8, 1 358.6±559.2 vs 625.6±336.3), and the differences were statistically significant (t=4.30, 10.60 and 3.50, all P<0.05); however, the expressions of IL-4 and TGF-β at mRNA levels significantly up-regulated (0.3±0.2 vs 0.6±0.1 and 0.4±0.1 vs 0.5±0.1), and the differences were statistically significant (t=5.20 and 12.50, P<0.05). Conclusions The regulation effects of 6-thiopurine on RIP3 signaling pathway and related cytokines are similar to those of Necrostatin-1. And the expression of RIP3 signaling protein increasing in activated macrophages of liver tissues from AIH patients is closely related to the regulation of IL-6. The RIP3-mediated inflammatory signaling pathway in macrophage may be involved in the genesis and development of AIH and may be a potential therapeutic target. Key words: Hepatitis, autoimmune; Macrophages; Receptor interacting protein serine threonine kinase 3; Interleukin-6

Effect of dexmedetomidine on mTOR/tau protein signaling pathway in hippocampus of aged rats after splenectomy

Objective To evaluate the effect of dexmedetomidine on the mammalian target of rapamycin(mTOR)/tau protein signaling pathway in the hippocampus of aged rats after splenectomy. Methods One hundred and fifty pathogen-free healthy male Sprague-Dawley rats, aged 18 months, weighing 400-540 g, were divided into 5 groups(n=30 each)using a random number table: control group(group C), sham operation group(group S), operation group(group O), normal saline group(group NS)and dexmedetomidine group(group D). Group C received no treatment.Ten percent chloral hydrate 0.3 ml/100 g was injected intraperitoneally in group S. Group O underwent splenectomy.Dexmedetomidine 50 μg/kg was injected intraperitoneally at 5 min before splenectomy in group D. The equal volume of normal saline was injected intraperitoneally at 5 min before splenectomy in group NS.Morris water maze test was performed at day 7 after surgery.At days 1, 3 and 7 after surgery, the rats were sacrificed, and the hippocampi were removed for examination of the pathological changes in the hippocampal CA3 region and for determination of the expression of mTOR protein and mRNA, tau protein mRNA and phosphor-tau protein(pS396 tau protein)(by real-time polymerase chain reaction or Western blot). Results Compared with group C, the escape latency and swimming distance were significantly prolonged, and the expression of mTOR protein and mRNA, tau protein mRNA and pS396 tau protein was up-regulated in O, D and NS groups(P 0.05). Compared with group O, the escape latency and swimming distance were significantly shortened, and the expression of mTOR protein and mRNA, tau protein mRNA and pS396 tau protein was down-regulated in group D(P 0.05). The pathological changes in the hippocampal CA3 region were significantly attenuated in group D as compared with group O. Conclusion The mechanism by which dexmedetomidine improves postoperative cognitive function may be associated with inhibited activation of mTOR/tau protein signaling pathway in the hippocampus of aged rats. Key words: Dexmedetomidine; Receptor-interacting protein serine-threonine kinases; tau proteins; Aged; Cognition disorders; Postoperative complications

Role of mTOR signaling pathway in dexmedetomidine-induced reduction of renal ischemia-reperfusion injury in rats: the relationship with HIF-1α

Objective To evaluate the role of mammalian target of rapamycin (mTOR) signaling pathway in dexmedetomidine-induced reduction of renal ischemia-reperfusion (I/R) injury in rats and the relationship with hypoxia-inducible factor 1 (HIF-1α). Methods Seventy-two male Sprague-Dawley rats, aged 10–12 weeks, weighing 220–260 g, were randomly divided into 4 groups (n=18 each) using a random number table: sham operation group (group S), group I/R, dexmedetomidine group (group Dex), and rapamicyn + dexmedetomidine group (group Rpm+ Dex). Renal I/R was produced by occlusion of bilateral renal pedicles for 35 min follow by reperfusion in anesthetized rats in I/R, Dex and Rpm+ Dex groups.Bilateral renal pedicles were only exposed, and then the abdominal cavity was closed in group S. Dexdetomidine 50 μg/kg was injected intraperitoneally at 30 min before I/R in group Dex.In group Rpm+ Dex, rapamicyn 1.5 mg/kg and dexdetomidine 50 μg/kg were injected intraperitoneally, and renal I/R model was established 30 min later.Immediately after onset of reperfusion, and at 4 and 24 h of reperfusion (T1-3), blood samples were collected from the caudal vein for measurement of serum creatinine and blood urea nitrogen (BUN) concentrations.After blood sampling at T1-3, the rats were sacrificed, and the renal specimens were obtained for detection of HIF-1α, erythropoietin (EPO) and mTOR expression by Western blot.Their kidneys were removed at T3, and cut into sections which were stained with haematoxylin and eosin and examined under microscope.Acute renal tubular necrosis was scored.The cell apoptosis in renal tissues was detected by TUNEL assay, and apoptosis index (AI) was calculated. Results Compared with group S, the concentrations of serum creatinine and BUN, expression of HIF-1α, EPO and mTOR at T2, 3, AI at T3 and acute renal tubular necrosis score were significantly increased in the other three groups (P 0.05). Conclusion The mTOR signaling pathway is involved in dexmedetomidine-induced reduction of renal I/R injury, which may be related to dexmedetomidine-produced up-regulation of HIF-1α expression in renal tissues of rats. Key words: Sirolimus; Dexmedetomidine; Receptor-interacting protein serine-threonine kinases; Hypoxia-inducible factor 1, alpha subunit; Kidney; Reperfusion injury

Role of hippocampal mTOR signaling pathways in cognitive dysfunction after splenectomy in aged rats

Objective To evaluate the role of hippocampal mammalian target of rapamycin(mTOR)signaling pathways in the cognitive dysfunction after splenectomy in aged rats. Methods Sixty-three male Sprague-Dawley rats, aged 18 months, weighing 400-540 g, were randomly divided into 3 groups(n=21 each)using a random number table: control group(group C), operation group(group O)and mTOR inhibitor rapamycin group(group M). Morris water maze test was performed to evaluate cognitive function before operation and at 1, 3 and 7 days after operation.After the end of Morris water maze test carried out at 1, 3 and 7 days after operation, 7 rats selected randomly in each group were sacrificed, and the brains were removed for detection of the expression of mTOR and phosphorylated tau protein at Ser-396 site(pS396 tau)in hippocampal tissues by using Western blot. Results Compared with group C, the escape latency was significantly prolonged, the ratio of time spending in the target quadrant was decreased, and the expression of pS396 tau was up-regulated at 1 and 3 days after operation, and the expression of mTOR was up-regulated at each time points after operation in group O, and the escape latency was significantly prolonged, the ratio of time spending in the target quadrant was decreased, and the expression of mTOR and pS396 tau was up-regulated at 1 and 3 days after operation in group M. Compared with group O, the escape latency was significantly shorten, the ratio of time spending in the target quadrant was increased, and the expression of pS96 tau was down-regulated at 1 and 3 days after operation, and the expression of mTOR was down-regulated at each time point after operation in group M. Conclusion The hippocampal mTOR signaling pathways are involved in the development of cognitive dysfunction after splenectomy in aged rats. Key words: Receptor-interacting protein serine-threonine kinases; Hippocampus; Cognition disorders; Postoperative complications; Aged

Role of hippocampal mammalian target of rapamycin signaling pathway in inflammatory pain in rats

Objective To evaluate the role of hippocampal mammalian target of rapamycin (mTOR) signaling pathway in inflammatory pain in rats. Methods Sixty adult male Sprague-Dawley rats, weighing 180-240 g, were randomly divided into 4 groups (n=6 each) by using a random number table: control group (group C), inflammatory pain group (group IP), dimethyl sulfoxide (DMSO) group and rapamycin (inhibitor of mTOR) group (group R). Inflammatory pain was produced by injection of honey bee venom 50 μl into the plantar surface of the left hindpaw.While the equal volume of normal saline was given instead in group C. In group D, 2% DMSO was injected through a gastric tube into stomach 1 ml per day lasting for 3 days, and inflammatory pain was produced at 1 h after the last injection on 3rd day.In group R, rapamycin 10 mg/kg (in 2% DMSO) was injected through a gastric tube into stomach 1 ml per day lasting for 3 days, and inflammatory pain was produced at 1 h after the last injection on 3rd day.At 2 h after the model was established, the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured.Rats were sacrificed after measurement of pain threshold, and hippocampal tissues were obtained for detection of the expression of mTOR, phosphorylated mTOR (p-mTOR), ribosomal S6 kinase (S6K) and phosphorylated S6K (p-S6K). Results Compared to group C, the MWT was significantly decreased, TWL was shortened, the expression of p-mTOR and p-S6K was up-regulated, and no significant change was found in the expression of mTOR and S6K in IP and DMSO groups, and no significant change was found in group R in the MWT, TWL and expression of p-mTOR, mTOR, p-S6K and S6K.Compared to group IP, no significant change was found in group DMSO in the MWT, TWL and expression of p-mTOR, mTOR, p-S6K and S6K, and the MWT was significantly increased, TWL was prolonged, the expression of p-mTOR and p-S6K was down-regulated, and no significant change was found in the expression of mTOR and S6K in group R. Conclusion Hippocampal mTOR signaling pathways are involved in the development of inflammatory pain in rats. Key words: Receptor-interacting protein serine-threonine kinases; Hippocampus; Pain; Inflammation

Role of mammlian target of rapamycin signaling pathway in spinal cord in development of neuropathic pain in rats

Objective To investigate the role of mammlian target of rapamycin (mTOR) signaling pathway in the spinal cord in the development of neuropathic pain (NP) in rats.Methods Twenty adult male SpragueDawley rats,weighing 180-220 g,were randomly divided into d groups (n =5 each) using a random number table:control group (group C),sham operation group (group S),NP group,and rapamycin group (group R).Intrathecal catheters were successfully implanted in the rats except those in group C and observed for 3 days.In group S,the left sciatic nerve was only exposed but not ligated.NP was produced by clamping the left sciatic nerve.In group R,rapamycin 10 μl was injected intrathecally at 4 h after NP for 3 consecutive days.At 1 day before operation and 3,5 and 7 days after NP,the mechanical pain threshold (MPT) was measured and the changes in synaptic structure and organelles in the 1amine Ⅱ of the spinal dorsal horn were studied with transmission electron microscope and stereological analysis.Results Compared with C and S groups,MPT was significantly decreased,the numerical density of all synapses,spine synapses,negative synapses and perforated synapses,thickness of the postsynaptic density and curvature of the synaptic interface were increased at 5 and 7 days after ligation in NP and R groups (P ＜ 0.05 or 0.01).Compared with NP group,MPT was significantly increased,the numerical density of all synapses,spine synapses,negative synapses and perforated synapses,thickness of the postsynaptic density and curvature of the synaptic interface were decreased at 5 and 7 days after ligation in R group (P ＜ 0.05).Conclusion mTOR signaling pathway in the spinal cord is involved in the development of NP in rats possibility through regulating synaptic plasticity in the spinal dorsal horn. Key words: Spinal cord; Receptor-interacting protein serine-threonine kinases; Neuralgia

Regulatory role of acetylcholine receptor in muramyl dipeptide-induced activation of NLR2/RIP2 pathway in macrophages of mice

Objective To evaluate the regulatory role of acetylcholine receptor in muramyl dipeptide (MDP)-induced activation of Nod-like receptor 2/receptor-interacting protein 2 (2NLR2/RIP2) pathway in macrophages of mice.Methods RAW264.7 cells at the logarithmic growth phase were seeded in 12-well plates (density 1 × 106 cells/ml,2 ml/well),a total of 108 wells.The cells were randomly divided into 3 groups (n =36 each) using a random number table:control group (group C),MDP group (group M),and GTS-21 (a7nAChR specific agonist) group (group G).The cells were routinely cultured in group C.MDP with the final concentration of 10 μg/ml was added to the culture medium in group M.MDP with the final concentration of 10μg/ml and GTS21 with the final concentration of 50 μg/ml were added to the culture medium in group G.The cells were incubated for 24 h.At 1,6 and 24 h of incubation with MDP,12 wells were chosen and the cell suspension was obtained for measurement of NLR2 mRNA expression (by real-time fluorescent quantitative PCR),RIP2 expression (by Western blot),and concentrations of tumor necrosis factor-alpha (TNF-α) and high mobility group box-1 (HMGB1) in the culture media (by ELISA).Results Compared with group C,the levels of NLR2 mRNA,RIP2,TNFα and HMGB1 were significantly increased at each time point in group M (P ＜ 0.05).Compared with group M,the levels of NLR2 mRNA,RIP2,TNF-α and HMGB1 were significantly decreased at each time point in group G (P ＜ 0.05).Conclusion Acetylcholine receptor can suppress MDP-induced transduction of NLR2/RIP2 pathway in macrophages of mice. Key words: Cholinergic agonists; Macrophages ; Acetylmuramyl-alanyl-isoglutamine ; Nod2 signaling adaptor protein; Receptor-interacting protein serine-threonine kinase 2

Role of mTOR in spinal cord in development of diabetic neuropathic pain in rats

Objective To evaluate the role of mTOR in spinal cord in the development of diabetic neuropathic pain in rats.Methods Sixty adult male Sprague-Dawley rats,aged 2 months,weighing 180-220 g,were used in the study.Forty-five rats among them were chosen randomly and diabetes mellitus was induced by intraperitoneal streptozotocin (STZ) 60 mg/kg and confirmed by blood glucose ＞ 16.7 mmol/L on day 3 after STZ injection.The left 15 rats received intraperitoneal injection of the equal volume of citric acid-sodium citrate buffer and served as normal control group (group C).Paw withdrawal threshold to von Frey filament stimulation was measured in the right hind paw before STZ injection and on 3,6,9,12,15,18,and 21 days after STZ injection.The diabetic rats with mechanical pain threshold decreasing by more than 50％ of the baseline were allocated to diabetic neuropathic pain group (group DP),and by less than 25 ％ of the baseline were allocated to diabetic non-neuropathic pain group (group NP).The rats were sacrificed at 21 days after STZ injection,and their lumbar enlargements of the spinal cord were removed for determination of the expression of mTOR and phosphorylated mTOR (p-mTOR) by Western blot.Results The expression of mTOR was significantly up-regulated in DP and NP groups when compared with group C (P ＜ 0.05),the expression of p-mTOR was up-regulated in DP group,and no significant change was found in the expression of p-mTOR in group NP (P ＞ 0.05).Compared with group NP,the expression of p-mTOR was significantly up-regulated (P ＜ 0.05),and no significant change was found in the expression of mTOR in group DP (P ＞ 0.05).Conclusion Activation of mTOR in the spinal cord is involved in the development of diabetic neuropathic pain in rats. Key words: Receptor-interacting protein serine-threonine kinases; Diabetic neuropathies

Tumor necrosis factor-mediated cell death: to break or to burst, that's the question.

In this review, we discuss the signal-transduction pathways of three major cellular responses induced by tumor necrosis factor (TNF): cell survival through NF-kappaB activation, apoptosis, and necrosis. Recruitment and activation of caspases plays a crucial role in the initiation and execution of TNF-induced apoptosis. However, experimental inhibition of caspases reveals an alternative cell death pathway, namely necrosis, also called necroptosis, suggesting that caspases actively suppress the latter outcome. TNF-induced necrotic cell death crucially depends on the kinase activity of receptor interacting protein serine-threonine kinase 1 (RIP1) and RIP3. It was recently demonstrated that ubiquitination of RIP1 determines whether it will function as a pro-survival or pro-cell death molecule. Deeper insight into the mechanisms that control the molecular switches between cell survival and cell death will help us to understand why TNF can exert so many different biological functions in the etiology and pathogenesis of human diseases.