Effect of JNK/MCP-1 signaling pathway on anti-diabetic neuropathic pain by curcumin in type 2 diabetic rats

AIM:To investigate the effect of JNK/MCP-1 signaling pathway on anti-diabetic neuropathic pain by curcumin in type 2 diabetic rats. METHODS:The male Sprague-Dawley rats were induced as the model of the type 2 diabetic neuropathic pain rats, they were randomly divided into 6 groups (n=27): type 2 diabetic neuropathic pain (DNP) group, type 2 diabetic neuropathic pain and intraperitoneal injection of curcumin (Cur) group, type 2 diabetic neuropathic pain and solvent control (DSC) group, type 2 diabetic neuropathic pain and JNK inhibitor (DJ) group, type 2 diabetic neuropathic pain and JNK inhibitor solvent control (DJS) group, type 2 diabetic neuropathic pain and monocyte chemoattractant protein 1 (MCP-1) agonist (DM) group. Another 27 normal SD rats were selected as control group. Mechanical withdrawal threshod and thermal withdrawal latency were measured at 3rd d, 7th d and 14th d after dosing, then the lumbar segment 4~6 of the spinal cord and L4~6 DRG were removed at the same time. ELISA was used to measure MCP-1 level. The expression of p-JNK was determined by Western blotting. RESULTS:Compared with DNP group, p-JNK was significantly decreased at 7th d and 14th d in Cur group, DJ group and DM group after treatment (P<0.05). Compared with C group, the MCP-1 was significantly declined in other 6 group after streptozotocin injection (P<0.05). Compared with DNP group, MCP-1 were significantly increased at 7th d and 14th d in Cur group and DJ group after treatment (P<0.05), and that in DM group was greatly decreased (P<0.05). CONCLUSION: The expression of p-JNK and MCP-1 was increased in DNP rats with spinal cord and dorsal root ganglion. The mechanism of curcumin reducing the neuropathic pain in type 2 diabetic rats might be through regulating the JNK/MCP-1 pathway.     

Dexmedetomidine reduced isoflurane-induced neuroapoptosis by inhibiting activation of p38 and JNK proteins in hippocampus of neonatal rats

AIM：To investigate the effect of dexmedetomidine (Dex) on neuronal apoptosis induced by isoflurane (Iso) and its relationship with the expression of p38 mitogen-activated protein kinase (p38) and c-Jun N-terminal kinase (JNK) proteins in the hippocampus of neonatal rats. METHODS：Forty-eight neonatal SD rats at postnatal day 7 were randomly divided into control group (Con), Dex group, Iso group and Iso combined with Dex (Iso+Dex) group. Rats in Iso and Iso+Dex groups were exposed to 0.75% Iso for 6 h, while rats in Con and Dex groups were exposed to air for 6 h. Rats were intraperitoneally injected with 25 μg·kg-1 Dex (Dex and Iso+Dex groups) or 150 μL saline (Con and Iso groups) 20 min before exposure and 2 and 4 h after exposure. After the termination of anesthesia, the neuronal apoptosis in hippocampal CA1 region was detected by TUNEL staining, and the protein expression of cleaved caspase-3, phospho-p38 (p-p38), p38, phospho-JNK (p-JNK) and JNK in hippocampal tissues was detected by Western blotting. RESULTS：The number of TUNEL positive cells in hippocampal CA1 region of the rats in Iso group was increased by 447.57% (P<0.01) compared with Con group, while Dex significantly inhibited the increased TUNEL positive cells in Iso group by 75.18% (P<0.01). The expression of cleaved caspase-3 protein in Iso group was increased by 126.29% (P<0.01) compared with Con group, while Dex reversed the increased cleaved caspase-3 protein expression (P<0.01). Iso significantly increased the phosphorylation of p38 and JNK proteins (P<0.01), while Dex reversed the increased p-p38 and p-JNK proteins (P<0.01). CONCLUSION：Dex attenuates Iso-induced neuroapoptosis in the hippocampus of neonatal rats through inhibiting the phosphorylation of p38 and JNK proteins.     

Relationship between expression of PGP9.5 and clinicopathological features of cervical carcinoma,and in vitro study of underlying mechanism

AIM: To detect the protein expression of protein gene product(PGP9.5) in cervical carcinoma samples, and to explore its relationship with clinicopathological features and prognosis of cervical carcinoma patients. The potential value of PGP9.5-siRNA in the treatment of cervical cancer was preliminarily investigated in vitro. METHODS: The clinical data of cervical cancer patients(n=180) who received surgical treatment in Department of Gynecology of Tianjin Fifth Central Hospital and Tianjin Central Hospital of Gynecology and Obstetrics from January 2008 to June 2015 were retrospectively analyzed. Immunohistochemical staining for PGP9.5 in all pathological specimens from cervical cancer biopsy was performed. The patients were divided into high expression of PGP9.5 group and low expression of PGP9.5 group. The relationship between PGP9.5 expression and clinicopathological parameters, such as age, HPV infection, pathological grade, tumor diameter, lymph node metastasis, depth of invasion and clinical stage, were analyzed. The overall survival was analyzed by Kaplan-Meier method and the log-rank test. The PGP9.5-siRNA, NC-siRNA, PGP9.5 eukaryotic expression plasmid and empty vector were transfected into the SiHa cells, and the effects of PGP9.5 expression on the abilities of colony formation and cell invasion were determined by Western blot, colony formation assay and Transwell experiment. RESULTS: The relationships between the expression level of PGP9.5 and patients' pathological characteristics including grade, tumor size, lymph node metastasis, depth of invasion, vascular involvement and clinical stage were statistically significant(P<0.05). The overall survival rates of 3 and 5 years in PGP9.5 high expression group were significantly lower than those in PGP9.5 low expression group(P<0.05). Compared with control group, the protein expression of PGP9.5, the number of colony formation and the number of invasive cells in si-PGP9.5 group were significantly decreased. The protein expression of PGP9.5, the number of colony formation and the number of invasive cells in PGP9.5 group were significantly increased. CONCLUSION: Over-expression of PGP9.5 protein indicates poor prognosis of cervical cancer, which may be a good predictor for the prognosis of cervical cancer patients. Inhibition of PGP9.5 expression may be an effective way of gene therapy for cervical cancer.     

Effects of AT1 receptor autoantibody on renal cell apoptosis and JNK expression in diabetic nephropathy rats

AIM:To study the effects of angiotensin Ⅱ type 1 receptor autoantibody (AT1-AA) on the apoptosis of renal cell and the expression of c-Jun N-terminal kinase (JNK) in diabetic nephropathy (DN) rats. METHODS:High-sucrose and high-fat diet and intraperitoneal injection of streptozotocin were utilized to induce DN rat model. We employed enzyme-linked immunosorbent assay (ELISA) for serum AT1-AA and TUNEL staining for renal cell apoptosis. Furthermore, Western blotting was performed to measure the levels of endoplasmic reticulum stress (ERS) chaperone glucose-regulated protein 78 (GRP78) and ERS-associated apoptosis protein p-JNK. RESULTS:The renal cell apoptotic rate in DN group was significantly increased compared with NC group, and the apoptotic renal cells in AT1-AA positive DN rats were much greater than those in AT1-AA negative DN rats (P<0.05). The protein levels of GRP78 and p-JNK were significantly increased compared with NC group. GRP78 and p-JNK protein levels also significantly increased in AT1-AA positive DN rats compared with AT1-AA negative DN rats. CONCLUSION: AT1-AA activates ERS response and induces renal cell apoptosis via the JNK apoptotic pathway in the renal tissues of DN rats.     

Effect of SAPK/JNK signal pathway on neuronal apoptosis in cerebral infarction rats

AIM: To investigate the molecular mechanism of neuronal apoptosis by observing the changes of key proteins in SAPK/JNK and Bcl-2/Bax signal pathways after brain infarction. METHODS: The cortical infarction was induced by photochemistry, namely photothrombotic cortical injury (PCI). Thirty-six Sprague-Dawley rats were randomly divided into 2 groups: PCI group and sham-operated group. The ipsilesional cortex was harvested for histomorphometry and transmission electron microscopy 7 days after PCI. Some key proteins including p-JNK1, p-JNK2, p-c-Jun, p-ATF-2, total JNK1, total JNK2, Bcl-2 and Bax were detected by Western blotting analysis.RESULTS: The cortical infarction in rats was successfully induced by photochemistry. The apoptosis of neurons in cortex was more obvious in PCI group than that in sham-operated group 7 days after PCI. The levels of p-JNK1, p-JNK2, p-c-Jun and p-ATF-2 in PCI group were significantly higher than those in sham-operated group, whereas the ratio of Bcl-2/Bax was significantly lower(P<0.05). CONCLUSION: Apoptosis is a major contributor to neuronal loss induced by cerebral hypoxia-ischemia for a long period after cortical infarction. The process is related to some apoptotic proteins such as Bcl-2/Bax and the SAPK/JNK signal pathways activated by ischemic injury.     

All-trans retinoic acid attenuates blood-brain barrier injury induced by cerebral ischemia-reperfusion in rats through JNK/P38 MAPK signaling pathway

AIM: To investigate the effect of all-trans retinoic acid (ATRA) on blood-brain barrier after cerebral ischemia-reperfusion (CIR) injury in rats and its possible role mechanism.METHODS: Male SD rats were randomly divided into sham group, model (CIR) group and CIR+ATRA (10, 30 and 90 mg/kg) groups. The rat model of CIR injury was established by MCAO thread occlusion method. After ischemia for 1.5 h and reperfusion for 24 h, the neurological functional behavioral score, cerebral infarction volume, brain water content and Evans blue content were determined. The activity of matrix metalloprotein-9 (MMP-9) was measured by gelatin zymography. The protein levels of claudin-5, occludin, ZO-1, JNK, p-JNK, P38, p-P38 and MMP-9 in the brain tissues were determined by Western blot.RESULTS: Compared with CIR model group, ATRA at 30 mg/kg significantly improved neurological function, and decreased cerebral infarction volume, brain water content, Evans blue content and the degradation of tight junction proteins in ischemic area (P<0.01). The activity and protein expression of MMP-9 in ischemic brain tissue were decreased (P<0.01). The phosphorylation of JNK and P38 was inhibited and the protein levels of p-JNK and p-P38 were decreased (P<0.01).CONCLUSION: ATRA reduces the damage of brain tissue and the destruction of blood-brain barrier induced by CIR in rats. The protective effect may be related to inhibiting the activation of JNK/P38 MAPK signaling pathway and MMP-9.     

Influence of 8-week swimming on peripheral neuropathy in type 2 diabetic rats

AIM: To explore the influence of long-term swimming on peripheral neuropathy in type 2 diabetic rats. METHODS: Male Wistar rats were fed with a high-fat and high-fructose diet, and injected with streptozocin to establish a model of type 2 diabetes mellitus. The rats were randomly divided into 4 groups: blank control group (C group), exercise control group (CE group), diabetes mellitus group (DM group) and diabetes mellitus+exercise group (DME group). The rats in CE group and DME group received 8-week swimming training (6 d/week). The training time was 20, 30 and 45 min in the first 3 d,respectively, and then it increased to 60 min a day. Eight weeks later, the motor nerve conduction velocity (MNCV) and the levels of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and C-reactive protein (CRP) in sciatic nerve tissues of the rats were measured. The morphological changes of the sciatic nerve were also observed under light microscope. RESULTS: Compared with DM group, 8-week swimming obviously accelerated the MNCV (P<0.05), decreased the levels of TNF-α, IL-6 and CRP in DME group (but no significant difference, P>0.05). The obvious nerve injury in DM group was observed. However, the pathological change of the sciatic nerve in DME group was relieved. CONCLUSION: Eight-week swimming training significantly accelerates the MNCV, attenuates the nerve injury in diabetic rats and has protective effect on peripheral nerve, which may be correlated with relieving the inflammatory reaction.     

Oxidative stress-activated JNK-MAPK signaling pathway is involved in fluctuant high glucose-induced injury of hepatocytes

AIM: To explore the mechanisms of fluctuant high blood glucose-induced apoptosis of hepatocytes. METHODS: SD rats were randomly divided into normal control group (N), stable high blood glucose group (S), fluctuant high blood glucose group (F) and insulin group (I). Diabetic rats were induced by intraperitoneal injection of streptozotocin (65 mg/kg), and the fluctuant high blood glucose animal model was induced by intraperitoneal injection of ordinary insulin and glucose at different time points every day. The blood glucose fluctuation patterns of the animals in F group within 12 weeks were similar every day and no significant difference of the HbA1c concentration was observed compared with S group, indicating that the fluctuant hyperglycemia was successfully established in F group. The activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the content of malondialdehyde (MDA) and nitric oxide (NO) in the homogenate of the liver tissues were detected by colorimetry. The mRNA and protein levels of JNK, p-JNK, Bax and Bcl-2 were examined by RT-PCR and Western blot. RESULTS: After 12 weeks, the increases in the intakes of food and water, the urine output, and the abnormal liver function were observed in S group, I group and F group. Compared with N group, the MDA level was increased, the content of NO and the activity of SOD and GSH-Px were decreased, and up-regulation of JNK mRNA and p-JNK and Bax proteins, and down-regulation of Bcl-2 were also found in S group, I group and F group. The above effects were more obviously showed in F group. CONCLUSION: Oxidative stress activates JNK-MAPK signaling pathway, which is involved in fluctuant high glucose-induced apoptosis of hepatocytes.     

PSMA activates JNK/SAPK pathway and regulates apoptosis of prostate cancer cells

AIM：To investigate the effects of prostate-specific membrane antigen (PSMA) on the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) pathway and the apoptosis of prostate cancer cells. METHODS：The shRNA lentiviral vector with high efficiency was constructed in the previous study to block the PSMA expression in the prostate cancer cells as experimental interference group, while the constructed vector of PSMA was transfected into the prostate cancer cells to promote PSMA expression as positive experimental group. The control group was the cell line without any treatment. JNK/SAPK inhibitor SP600125 was used as a negative control. Western blotting and immunocytochemistry were used to observe the p-JNK/SAPK expression. The cell growth curve was determined by CCK-8 assay. The cell cycle and apoptosis were analyzed by flow cytometry. RESULTS：Inhibition of PSMA expression resulted in the decrease of p-JNK/SAPK expression levels, while enhancement of the PSMA expression made the increase in the expression of p-JNK/SAPK. SP600125 decreased the level of p-JNK/SAPK, and no significant difference among the 3 groups was observed. The cell proliferation and S-phase percentage decreased after the inhibition of PSMA, while the cells in the 3 groups with SP600125 treatment only had low levels of cell proliferation and percentage of S phase. The inhibition of PSMA promoted apoptosis, while in the enhanced PSMA expression group, apoptotic rate was significantly reduced. After adding SP600125, the cell apoptotic rate was lower than that in normal culture group. CONCLUSION：PSMA has an impact on the proliferation, cell cycle and apoptosis of prostate cancer cells by up-regulating JNK/SAPK signaling pathway, but the JNK/SAPK signaling is not the only path.     

Protective effect of curcumin analogue L6H4 on diaphragm of type 2 diabetic rats

AIM: To investigate the protective effect of curcumin analogue L6H4 on diaphragm of type 2 diabetic rats.METHODS: SPF male Sprague-Dawley rats (n=40) were randomly divided into 5 groups: normal control (NC) group, high fat (HF) group, high fat+L6H4 treatment (FT) group, diabetes mellitus (DM) group and DM+L6H4 treatment (DT) group. The rats in the later 4 groups were fed with high-fat diet. After 4 weeks of high-fat diet fee-ding, the rats in DM and DT groups were intraperitoneally injected with streptozotocin to induce type 2 diabetes melliutus. The rats in FT and DT groups were given L6H4 by gavage for 8 weeks. Blood glucose and blood lipid levels were detected biochemically. Fasting serum insulin (FINS) level was measured by radioimmunoassay and insulin resistance index (HOMA-IR) was calculated. Serum adiponectin (APN) level was measured by ELISA. The morphological changes of the diaphragm were observed under light and transmission electron microscopes. Lipid deposition and the activity of succinate dehydrogenase (SDH) and NADH-tetrazolium reductase (NADH-TR) were observed by enzyme histochemical staining. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in the diaphragm were measured by thiobarbituric acid method and hydroxylamine method, respectively. The protein expression of adiponectin receptor 1 (AdipoR1) in the diaphragm was determined by immunohistochemistry and Western blot. RESULTS: The levels of blood lipids, blood glucose, FINS and HOMA-IR in HF and DM groups were higher than those in NC group, but decreased after L6H4 treatment. The serum APN level in HF and DM groups was lower than that in NC group, but increased after treatment with L6H4. The muscle fibers of the diaphragm were shrunk, fat particles accumulated in the muscle fibers, and the mitochondria were slightly swollen in HF and DM groups. The diaphragmatic fibrosis was obvious in DM group. These lesions were relieved after L6H4 treatment. Compared with NC group, the level of MDA and the activity of SDH and NADH-TR in the diaphragm were increased in HF and DM groups, but decreased after treatment with L6H4. The activity of SOD and the expression of AdipoR1 in the diaphragm were lower than those in NC group, but increased after L6H4 treatment.CONCLUSION: The curcumin analogue L6H4 exerts a protective effect on diaphragm in type 2 diabetic rats. The strengthened protein expression of AdipoR1, the increased serum level of APN, and anti-lipid peroxidation may be involved in the process.     

Effect of NAC on oxidant stress and apoptosis in the hippocampal CA1 region of rats exposured to chronic intermittent hypoxia

AIM: To investigate the effect of N-acetylcystein (NAC) on oxidant stress, neuron apoptosis in the hippocampal CA1 region of rats exposured to chronic intermittent hypoxia (CIH). METHODS: 30 healthy male Wistar rats were randomly divided into three groups of 10 each, a CIH group, a NAC therapeutic group and a control group. The levels of MDA and SOD were detected by colorimetric method. Immunohistochemistry was used to examine the expression of p-JNK and TUNEL was used to detect the neuron apoptosis in the hippocampal CA1 region. RESULTS: The level of MDA in NAC group were lower than that in CIH group［(1.71±0.43) μmol/g protein vs （1.37±0.26) μmol/g protein, P<0.05)］. The activity of SOD in NAC group was higher than that in CIH group［(44.94±14.01) 103 NU/g protein vs(57.66±14.07) 103 NU/g protein, P<0.05)］. The expression of p-JNK protein and the apoptotic indices ［(0.39±0.16), (0.20±0.11)］ in NAC group were significantly lower than those in CIH group ［(0.53±0.10), (0.32±0.18), all P<0.05］. CONCLUSION: NAC protects hippocampal neuron from apoptosis by suppressing the oxidant stress in the hippocampal CA1 region and inhibiting the activation of JNK signaling pathway.     

Anti-aging effect of stromal vascular fractions in photoaging skin

AIM: To investigate the anti-aging effect of the stromal vascular fractions (SVF) injection in photo-aging skin of nude rats. METHODS: Six-week-old nude rats were divided into control group and photoaging group. The nude rats in photoaging group were continuously irradiated with ultraviolet B (UVB) for 8 weeks to build the photoaging skin model, and were randomly divided into placebo group, and low-dose (LD), middle-dose (MD) and high-dose (HD) SVF treatment groups. The human SVF was obtained by trypsin digestion from the adipose tissue in healthy females, and was subcutaneously injected into the back skin of photoaging nude rats in LD, MD and HD SVF treatment groups (10⁴,10⁵ and 10⁶ cells in 100 μL suspension, respectively). The animals in photoaging skin model group were treated with placebo. Control group did not have any intervention. The changes in the thickness of epidermis and histology after 7 d and the thickness of the dermis and histology after 28 d were examined. RESULTS: The thickness of the epidermis was thinner in MD and HD SVF treatment groups than that in photoaging skin model group after 7 d (P<0.05). The percentage of stratum corneum and the abnormal proliferation of the epidermal basal cell layer were reduced (P<0.05). After 28 d, MD and HD SVF treatment groups showed that the content of collagen I was higher than that in photoaging skin model group (P<0.05), and the contents of collagen III and MMP3 were decreased (P<0.05). The dermis in HD group was thicker than that in photoaging skin model group (P<0.05).CONCLUSION: SVF may have anti-aging potential in photoaging skin and also has clinical application value.     

Effects of curcumin analogue L6H4 on myocardial tissue of type 2 diabetic rats

AIM: To explore the effects of curcumin analogue L6H4 on the myocardial tissue of type 2 diabetic rats and its mechanism. METHODS: Male Sprague-Dawley rats were randomly divided into normal control (NC) group, high-fat (HF) group, high-fat treatment (FT) group, diabetes mellitus (DM) group and diabetes treatment (DT) group.The rats in the latter 4 groups were fed high-fat diet for 4 weeks, then the rats in DM groups and DT groups were intraperitoneally injected with streptozotocin (STZ) to induce type 2 diabetes, while the rats in FT group and DT group were given L6H4. The blood glucose and lipid levels were detected by biochemical method, and serum adiponectin (APN) levels were detected by ELISA. The serum insulin levels were measured by radioimmunoassay and homeostasis model assessment of insulin resistance (HOMA-IR) were calculated. The morphological changes of myocardium were observed by Masson staining and electron microscopy. The protein expression of adiponectin receptor 1 (AdipoR1) and transforming growth factor β1(TGF-β1) in myocardial tissue were determined by immunohistochemistry. The protein expression of adipoR1 was also detected by Western blot for verification. RESULTS: Compared with NC group, the blood glucose, lipids, insulin, HOMA-IR and TGF-β1 were increased in HF and DM group, but they were decreased after treated with L6H4. Compared with NC group, the concentration of serum APN were decreased and the expression of AdipoR1 in the myocardium were weakened in HF group and DM group, and they increased after treated with L6H4. The myocardial fibrosis was obvious in HF group and DM group, the mitochondria in cardiomyocytes expanded, and the cristae disordered, partial disappeared. These lesions were significantly reduced after L6H4 treatment. CONCLUSION: L6H4 exerts a protective effect on the heart in type 2 diabetic rats. The increased concentration of serum APN, the enhanced expression of AdipoR1, and the expression of TGF-β1 inhibited by APN may be involved in the mechanism of protection.     

Effects of isoflurane and sevoflurane on apoptosis of cortical neuron in neonatal rats and the role of MAPKs pathway

AIM: To investigate the effects of isoflurane and sevoflurane at the same dose on apoptosis of cortical neuron in neonatal rats and the role of mitogen-activated protein kinases (MAPKs) pathway.METHODS: Eleven neonatal rats were selected at postnatal day 7 from 1 litter (altogether 5 litters) and assigned randomly into control group (C group), isoflurane group (Ⅰ group) and sevoflurane group (S group). The rats in Ⅰ group, S group or C group were exposed to 1.1% isoflurane, 1.8% sevoflurane and room air for 4 h, respectively. The brain of neonatal rats were perfused and embedded by paraffin. Caspase-3 positive expression in the retrosplenial cortex (RS) of the brain was observed by immunohistochemical staining. Meanwhile, the fresh cortex was separated at 0 h in C group and at 2 h and 4 h in Ⅰ group and S group. The levels of phospho-SAPK/JNK and SAPK/JNK, phospho-p38 and p38 in fresh cortex were detected by Western blotting.RESULTS: Caspase-3 positive cells in the the cortex were increased by 441% in Ⅰ group (P<0.01) and 151% in S group (P<0.01) as compared to C group, and increased by 115% in Ⅰ group (P<0.05) as compared to S group. The protein levels of phospho-SAPK/JNK in the cortex were increased by 219% at 2 h (P<0.05) and 181% at 4 h (P<0.05) in Ⅰ group, while no significant difference between S group and C group was observed. The phospho-p38 protein in the cortex was increased by 38.9% at 2 h (P<0.05) and 36.9% at 4 h (P<0.05) in Ⅰ group, and increased by 32.6% (P<0.05) at 2 h and 128.0% at 4 h (P<0.01) in S group as compared to C group.CONCLUSION: Isoflurane induces more apoptotic neurons in the cortex of the brain in neonatal rats at postnatal day 7 than sevoflurane. Isoflurane induces apoptosis mainly by activating SAPK/JNK phosphorylation, while sevoflurane induces aopotosis by activating p38 phosphorylation.     

SHP-2 regulates PC12 cell survival and apoptosis in response to NGF via activation of ERK and JNK

AIM: To investigate the central role of the protein tyrosine phosphatase SHP-2 in the survival of PC12 cells upon NGF treatment and apoptosis after NGF withdrawal. METHODS: PC12 cells were treated with SHP-2 inhibitor (NSC87877). Cell survival rate was detected by MTT method and apoptosis was determined by flow cytometry. Moreover, pIRES-GFP (vector alone), pIRES-GFP-SHP-2 (wild type) and pIRES-GFP-SHP-2C459S (dominant negative mutant form) were transfected into PC12 cells. ERK, p-ERK, JNK and p-JNK were immunoblotted at 1 h in the presence of NGF and 5 h after NGF withdrawal. RESULTS: SHP-2 potentially enhanced survival and attenuated apoptosis of PC12 cells. The activation of ERK was significantly enhanced with NGF treatment either in the group without SHP-2 inhibitor or the SHP-2 wild type group. On the other hand, phosphorylation level of JNK was significantly increased after NGF withdrawal when PC12 cells were treated with SHP-2 inhibitor or transfected with SHP-2 mutant. CONCLUSION: SHP-2 may play a central role in mediating the survival and apoptosis of PC12 cells upon NGF exposure and withdrawal. The underlying mechanisms may be through the activation of ERK and JNK.     

Expression of genes in MAPKs pathway in development of neural tube defects induced by hyperthermia

AIM: To study the expression and the role of ERK1/2 and JNK1/2 of MAPKs pathways in the development of neural tube defects induced by hyperthermia. METHODS: The animal models of golden hamster were produced by hyperthermia. The expression of ERK1/2 and JNK1/2, and levels of their phosphorylation were measured by Western blotting in control group and hyperthermia group. RESULTS: p-ERK1/2 steadily expressed in each control group, and the expression of p-ERK1/2 significantly decreased, which was different from that in the corresponding control group (P<0.05). The activity of p-JNK1/2 increased in hyperthermia group and the amount of p-JNK1/2 increased as compared to control group. The peak appeared at 16 h after exposed to hyperthermia (P<0.05). CONCLUSION: Hyperthermia, which induces a decrease in p-ERK1/2 expression and increases the expression of p-JNK1/2 of MAPKs pathway, results in the unbalance of cell proliferation and apoptosis, and induces neural tube defects.     

Effect of aerobic exercise on myenteric plexus and colonic dysfunction in type 2 diabetic rats

AIM: To observe the effect of aerobic exercise and dietary patterns on the colonic function of type 2 diabetic rats and the enteric nervous mechanism.METHODS: The rat model of type 2 diabetes was induced by high-fat diet (HFD) and streptozotocin (30 mg/kg, ip) injection, and the rats were divided into diabetes control (DC) group, HFD group, exercise (E) group and exercise combined with high fat diet (E+HFD) group. Some other healthy rats were arranged into normal control (NC) group. The rats in E group and E+HFD group received 8-week swimming training (5 d/week, 60 min/d). The colon samples were collected at the end of the 8th week for observation of the pathological changes by HE staining and for detection of colonic tension and expression of protein gene product 9.5(PGP9.5), substance P(SP) and vasoactive intestinal peptide (VIP).RESULTS: Diabetes induced significant myenteric plexus damages and marked reduction of neurons, while exercise protected the enteric nervous system from injuries. The expression of SP significantly increased in the rats with long-term aerobic exercise combined with a reasonable diet. However, high-fat diet combined with exercise did not obviously up-regulate SP. The positive expression of VIP in the colon significantly increased in both E group and E+HFD group. Aerobic exercise attenuated the atrophy and increased the tension in colonic smooth muscles.CONCLUSION: Diabetes induces muscular atrophy and tension attenuation in colonic smooth muscle, which can be reversed in some extent by aerobic exercise through the remolding of enteric nervous system.     

Preliminary mechanism of senegenin against H/R-induced apoptosis of primary cortical neurons

AIM：To explore the preliminary mechanism of senegenin (Sen) on inhibiting hypoxia/reoxygenation(H/R)-induced apoptosis of primary cortical neurons. METHODS：The cultured cortical neurons were randomly divided into normal group (control group), model group (H/R group), Sen+H/R group and Sen group. Flow cytometry was used to evaluate the effect of Sen on H/R-induced cell apoptosis. The protein levels of JNK, p-JNK, c-Jun, p-c-Jun, Bcl-2 and Bax were assessed by Western blotting. RESULTS：The apoptotic rate in H/R group was obviously higher than that in control group (P<0.05), while the apoptotic rate in Sen+H/R group was obviously lower than that in H/R group (P<0.05), suggesting that the model of apoptosis was established successfully. The results of Western blotting showed that Sen increased the expression of JNK and c-Jun, inhibited the phosphorylation of JNK and c-Jun (P<0.05), increased the protein level of Bcl-2 and inhibited the protein level of Bax in H/R treated primary cortical neurons (P<0.05). CONCLUSION：Sen has a protective effect against H/R-induced neuronal apoptosis by increasing the expression of JNK and c-Jun, inhibiting the phosphorylation of JNK and c-Jun, increasing the protein level of Bcl-2 and decreasing the protein level of Bax.