Effect of edaravone on ERK-CREB signaling pathway in hippocampus of aged rats with postoperative cognitive dysfunction / 中华麻醉学杂志

Objective:To evaluate the effect of edaravone on the extracellular signal-regulated kinase (ERK)-cAMP responsive element binding protein (CREB) signaling pathway in the hippocampus of aged rats with postoperative cognitive dysfunction (POCD).Methods:Sixty SPF healthy male Sprague-Dawley rats, aged 20 months, weighing 650-700 g, were divided into 4 groups ( n=15 each) using a random number table method: control group (group C), POCD group (group P), edaravone group (group E) and ERK inhibitor group (group I). The rats received laparotomy under 3% sevoflurane anesthesia to prepare POCD model in P, E and I groups. Edaravone 3 mg/kg was intraperitoneally injected at 30 min before operation in E and I groups, ERK inhibitor PD98059 0.3 mg/kg was injected via the tail vein in group I. The open field test was performed at 3 days after operation to evaluate the spontaneous activity of rats, then Morris water maze test was performed to evaluate the cognitive function of rats on 3-7 days after operation. The rats were sacrificed after the end of Morris water maze test, and hippocampal tissues were obtained for determination of the expression of phosphorylated ERK (p-ERK), phosphorylated CREB (p-CREB), synaptophysin and postsynaptic density protein 95 (PSD-95) (by Western blot) and dendrite length and density of dendrites in hippocampal CA1 area (using Golgi staining). Results:Compared with group C, the escape latency was significantly prolonged after operation, the number of crossing the original platform was reduced, the expression of p-ERK, p-CREB, synaptophysin and PSD-95 was down-regulated, and the dendritic length and density of hippocampal neurons were reduced in group P ( P<0.05). Compared with group P, the escape latency was significantly shortened, the number of crossing the original platform was increased, the expression of p-ERK, p-CREB, synaptophysin and PSD-95 was up-regulated, and the dendritic length and density of hippocampal neurons were increased in group E ( P<0.05). Compared with group E, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the expression of p-ERK, p-CREB, synaptophysin and PSD-95 was down-regulated, the dendritic length of hippocampal neurons was shortened, and the density of hippocampal neurons was decreased in group I( P<0.05). Conclusions:The mechanism by which edaravone improves POCD may be related to activating ERK/CREB signaling pathway and changing synaptic plasticity in hippocampal CA1 region in aged rats.

Thyroxine promotes the progression of integrin α vβ 3-positive differentiated thyroid cancer through the ERK1/2 pathway / 中华核医学与分子影像杂志

Objective:To explore whether thyroxine (T 4) could promote differentiated thyroid cancer (DTC) progression by binding to integrin α vβ 3in vitro and its downstream mechanism. Methods:Papillary thyroid cancer cell lines TPC-1, K1 and follicular thyroid cancer (FTC) cell line FTC133 were cultured in vitro, and the expressions of integrin α vβ 3 in those 3 DTC cell lines were determined with immunofluorescence and flow cytometry analysis. After the treatment of T 4, tetraiodo thyroacetic acid (Tetrac) and Arg-Gly-Asp (RGD) peptide alone or in combination, the proliferation and metastatic potential of DTC cell lines were detected by cell counting kit-8 (CCK-8), Transwell migration and invasion assays. The small interfering RNA (siRNA) transfection was used to verify whether integrin α v or β 3 subunit knockdown could reverse the effect of T 4 on DTC cells. The expression levels of downstream signaling proteins phosphorylated extracellular signal-regulated kinase (p-ERK)1/2 and total extracellular signal-regulated kinase (ERK)1/2 were detected by Western blot. The effects of mitogen-activated protein kinase kinase (MEK)1/2 inhibitor (GSK1120212) on the proliferation, migration and invasion of T 4-treated cells were detected. One-way analysis of variance and Tukey test were used for data analysis. Results:The integrin α vβ 3 expressions in TPC-1, K1 and FTC133 cells were all positive, with the relative mean fluorescence intensity (MFI) of 61.93±18.61, 16.89±2.43 and 32.36±0.83, and the percentages of positive cells of (94.38±1.30)%, (74.11±3.87)% and (50.67±1.78)%, respectively ( F values: 13.36 and 217.30, P=0.006 and P<0.001). Compared with control group, the proliferation, migration and invasion in the three DTC cell lines treated with T 4 were significantly enhanced (96 h, F values: 62.67-297.50, q values: 13.15-20.73, all P<0.001). T 4-induced cell proliferation, migration and invasion were markedly reversed by Tetrac or RGD (96 h, q values: 8.61-17.54, all P<0.001). T 4-induced cell proliferation, migration and invasion were also significantly inhibited by the knockdown of integrin α v or β 3 subunit (72 h, F values: 7.75-70.98, q values: 4.77-15.21, all P<0.05). Western blot results showed that the phosphorylation levels of ERK1/2 in DTC cells were significantly increased by T 4 treatment, and the T 4-induced activation of ERK1/2 signaling pathway could be blocked by Tetrac, RGD, integrin α v or β 3 subunit knockdown. T 4-induced cell proliferation, migration and invasion were significantly reversed by GSK1120212 (96 h, F values: 47.53-151.40, q values: 10.32-16.65, all P<0.001). Conclusion:T 4 can promote cell proliferation and metastasis of DTC cells by binding to integrin α vβ 3 and activating the ERK1/2 pathway.

Effect of rat cardiac fibroblasts on expression of Cx43 in H9c2 cells during hypothermic hypoxia/reoxygenation / 中华麻醉学杂志

Objective:To evaluate the effect of rat cardiac fibroblasts (RCF) on the expression of connexin43 (Cx43) in H9c2 cells during hypothermic hypoxia/reoxygenation.Methods:H9c2 cells cultured in vitro were divided into 4 groups ( n=12 each) using the random number table method: control group (group C), hypothermic hypoxia/reoxygenation group (group HHR), RCF co-culture group (group Co) and RCF co-culture plus hypothermic hypoxia/reoxygenation group (group Co+ HHR). Group C was incubated at 37℃ in 5% CO 2 + 95% air for 5 h. Group HHR was incubated at 4 ℃ in 5% CO 2 + 95% N 2 for 1 h and then at 37 ℃ in 5% CO 2 + 95% air for 4 h. In group Co and group Co+ HHR, H9c2 cells 0.3×10 5 cells/well were inoculated in the lower chamber and RCF 0.6×10 5 cells/well in the the upper chamber of a transwell ? culture dish.Group Co was incubated at 37 ℃ in 5% CO 2 + 95% air for 5 h. Group Co+ HHR was incubated at 4℃ in 95% N 2 + 5% CO 2 for 1 h, and then incubated at 37 ℃ in 5% CO 2 + 95% air for 4 h. The mortality rate of H9c2 cells was measured by trypan blue staining, the expression of Cx43 and extracellular signal-regulated protein kinases 1/2 (ERK1/2) by immunofluorescence, and the expression of Cx43, phosphorylated Cx43, ERK1/2 and phosphorylated ERK1/2 by Western blot. Results:Compared with group C, the mortality rate of H9c2 cells was significantly increased, the expression and phosphorylation of Cx43 were decreased, and the expression and phosphorylation of ERK1/2 were increased in group HHR ( P<0.05), and no significant change was found in the mortality rate of H9c2 cells or expression and phosphorylation of Cx43 and ERK1/2 in group Co ( P>0.05). Compared with group Co, the mortality rate of H9c2 cells was significantly increased, and the expression and phosphorylation of Cx43 and ERK1/2 were decreased in group Co+ HHR ( P<0.05). Compared with group HHR, the mortality rate of H9c2 cells was significantly increased, and the expression and phosphorylation of Cx43 and ERK1/2 were decreased in group Co+ HHR ( P<0.05). Conclusions:RCFs can decrease the expression and activity of Cx43 in H9c2 cells during hypothermic hypoxia/reoxygenation, and the mechanism may be related to the down-regulation of ERK1/2 expression and inhibition of ERK1/2 activity.

Preliminary study on the expression of MIF in HCC tissues and its relationship with ERK1/2 signaling pathway / 中华内科杂志

Objective:To investigate the expression of Macrophage migration-inhibitory factors (MIF) in hepatocellular carcinoma (HCC) tissues and its interaction with ERK1/2 signaling pathway, so as to establish a theoretical basis for further studying the molecular mechanism of MIF promoting HCC.Methods:From February 2020 to August 2021, 52 cases of hepatocellular carcinoma (HCC) tissues based on hepatitis B cirrhosis (HBV-LC) and 52 cases of adjacent tissues in Tianjin Medical University Cancer Hospital and 940th Hospital of Joint Logistic Support Force of PLA were collected as the experimental group, including 39 males and 13 females, aged 35-65 years. And 20 cases of normal liver tissue were selected as the control group. Immunohistochemistry was used to detect the expression of MIF, ERK1/2 and p-ERK1/2 proteins in liver tissues of the two groups, and in situ hybridization was used to detect the expression of ERK1/2 nucleic acid in liver tissues of the two groups.HepG2 HCC cells and L-02 normal hepatocytes were co-cultured with different concentrations of rMIF, the expression and phosphorylation levels of ERK1/2 and JNK1 proteins in the two kinds of liver cells were detected by Western-blot, and the expression levels of ERK1/2 nucleic acids in the two kinds of liver cells were detected by RT-PCR. One-way ANOVA was used for measurement data and χ 2 test was used for counting data. Results:The expressions of MIF, ERK1/2, p-ERK1/2 and ERK1/2 mRNA were significantly increased in HCC and para-cancer tissues (the expression of MIF in HCC group was 78.8%, and that in adjacent group was 75.0%; ERK1/2 80.8% in HCC group and ERK1/2 71.8% in paracancerous group. The expression of p-ERK1/2 75.0 % in HCC group and 46.2% in paracancerous group were respectively detected. ERK1/2 mRNA was expressed in HCC group 76.9%, ERK1/2 mRNA expression in paracancerous group 78.8%), and the differences were statistically significant compared with normal liver tissues ( P<0.05), but there was no significant difference between HCC and para-cancer tissues ( P>0.05). The expressions of ERK1/2, p-ERK1/2 and ERK1/2 mRNA in HepG2 HCC cells were significantly increased with the increase of rMIF concentration, and the increase was most obvious when rMIF concentration was 200 ng/ml, and the difference was statistically significant compared with L-02 normal hepatocytes ( P<0.05). Conclusion:MIF, ERK1/2 and p-ERK1/2 are highly expressed in HCC tissues and HepG2 HCC cells, suggesting that MIF promotes the occurrence and development of hepatocellular carcinoma through ERK1/2 signaling pathway.

Effect of remifentanil on MAPK signaling pathway during intestinal epithelial cell apoptosis induced by intestinal ischemia-reperfusion in rats / 中华麻醉学杂志

Objective:To evaluate the effect of remifentanil on mitogen-activated protein kinase (MAPK) signaling pathway during intestinal epithelial cell apoptosis induced by intestinal ischemia-reperfusion (I/R) in rats.Methods:Thirty-six clean grade healthy adult male Sprague-Dawley rats, weighing 200-250 g, aged 2 months, were divided into 3 groups ( n=12 each) by a random number table method: sham operation group (Sham group), intestinal I/R group (I/R group) and remifentanil group (R group). Intestinal I/R was produced by occlusion of superior mesenteric artery for 1 h followed by reperfusion in anesthetized rats.At 30 min before ischemia, 0.2 μg·kg -1·min -1 of remifentanil was infused intravenously for 5 min , followed by infusion of normal saline for 5 min, repeating for 3 cycles in group R. At 2 h of reperfusion, blood samples were collected from right ventricle to measure the concentration of diamine oxidase (DAO). The animals were then sacrificed and the intestinal tissues were obtained for examination of pathological changes and scored according to Chiu, for calculation of intestinal epithelial cell apoptosis rate (by TUNEL), for determination of the expression of phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated c-Jun N-terminal kinase (p-JNK), phosphorylated p38 MAPK (p-p38 MAPK), cleaved caspase-3 and nuclear factor kappa B p65 (NF-κB p65) in nucleoprotein and for calculation of p-ERK/ERK ratio, p-JNK/JNK ratio and p-p38 MAPK/p38 MAPK ratio in the intestinal tissues. Results:Compared with group Sham, Chiu′s scores, serum DAO concentration, apoptosis rate, p-ERK/ERK ratio, p-JNK/JNK ratio and p-p38 MAPK/p38 MAPK ratio and the expression of cleaved caspase-3 and NF-κB p65 in the intestinal tissues were significantly increased in group I/R, and Chiu′s scores was increased ( P<0.05), and no significant change was found in serum DAO concentration, apoptosis rate, p-ERK/ERK ratio, p-JNK/JNK ratio, p-p38 MAPK/p38 MAPK ratio and the expression of cleaved caspase-3 and NF-κB p65 in the intestinal tissues in group R ( P>0.05). Compared with group I/R, Chiu′s scores, apoptosis rate, serum DAO concentration, p-ERK/ERK ratio and expression of cleaved caspase-3 and NF-κB p65 were significantly decreased in group R ( P<0.05). Conclusion:The mechanism by which remifentanil inhibits intestinal epithelial cell apoptosis induced by intestinal I/R is related to promoting activation of ERK in rats.

Role of ERK signaling pathway in intrathecal dexmedetomidine-induced reduction of spinal cord ischemia-reperfusion injury in rats / 中华麻醉学杂志

Objective@#To evaluate the role of extracellular signal-regulated kinase (ERK) signaling pathway in intrathecal dexmedetomidine-induced reduction of spinal cord ischemia-reperfusion (I/R) injury in rats.@*Methods@#Eighty clean-grade male Sprague-Dawley rats, aged 9-10 weeks, weighing 300-350 g, were divided into 4 groups (n=20 each) using a random number table method: sham operation group (group S), spinal cord I/R group (group I/R), dexmedetomidine group (group D), and dexmedetomidine plus ERK signaling pathway blocker PD98059 group (group P). Spinal cord ischemia was produced by cross-clamping of the abdominal aorta distal to the left renal artery for 25 min followed by reperfusion to establish the model of spinal cord I/R injury.Dexmedetomidine 1 μg/kg was intrathecally injected at 20 min before establishing the model in D and P groups, PD98059 2 mg/kg was given via the tail vein at the same time in group P, and the equal volume of normal saline was given instead in S and I/R groups.Five rats were selected at 6, 8, 10 and 12 h of reperfusion, and the modified Basso, Beattie, Bresnahan (BBB) scale was used to assess the hindlimb locomotor function.Five rats were sacrificed after assessing the locomotor function at 6 h of reperfusion, and the L3-5 segments of the spinal cord were taken for determination of cell apoptosis (by TUNEL) and expression of phosphorylated ERK (p-ERK) (by Western blot). The apoptosis index was calculated.@*Results@#Compared with group S, the BBB scores were significantly decreased at each time point of reperfusion, the apoptosis index was increased, and the expression of p-ERK was up-regulated in the other three groups (P<0.05). Compared with I/R group, the BBB scores were significantly increased at each time point of reperfusion, the apoptosis index was increased, and the expression of p-ERK was up-regulated in group D, and no significant change was found in the apoptosis index or p-ERK expression in group P (P>0.05). Compared with group D, the BBB scores were significantly decreased at each time point of reperfusion, the apoptosis index was increased, and the expression of p-ERK was down-regulated in group P (P<0.05).@*Conclusion@#The mechanism by which intrathecal dexmedetomidine reduces spinal cord I/R injury is related to activating ERK signaling pathway in rats.

Role of GPR30 in 17β estradiol-induced inhibition of ketamine-caused neuroapoptosis in hippocampus of newborn rats: the relationship with p-ERK1/2 / 中华麻醉学杂志

Objective@#To evaluate the role of G protein-coupled receptor 30 (GPR30) in 17β estradiol-induced inhibition of ketamine-caused neuroapoptosis in the hippocampus of newborn rats and the relationship with phosphorylated extracellular signal-regulated kinase 1/2 (p-ERKl/2).@*Methods@#Twenty-four clean-grade healthy male Sprague-Dawley rats, aged 7 days, weighing 11-18 g, were divided into 4 groups (n=6 each) using a random number table method: control group (group C), ketamine group (group K), 17β estradiol plus ketamine group (group EK), and GPR30 inhibitor G15 plus 17β estradiol plus ketamine group (group G15EK). Ketamine 75 mg/kg (diluted to 0.1 ml in normal saline) was intraperitoneally injected every 24 h for 3 consecutive days in group K. In group EK, 17β estradiol 600 μg/kg was subcutaneously injected and ketamine 75 mg/kg was intraperitoneally injected every 24 h for 3 consecutive days.G15 300 μg/kg and 17β estradiol 600 μg/kg were subcutaneously injected and ketamine 75 mg/kg was intraperitoneally injected every 24 h for 3 consecutive days in group G15EK.The equal volume of normal saline 0.1 ml was intraperitoneally injected instead in group C. The animals were sacrificed at 24 h after the last injection for determination of the expression of cleaved caspase-3, ERK1/2 and phosphorylated ERK1/2(p-ERK1/2) (by Western blot).@*Results@#There was no significant difference in the expression of ERK1/2 in hippocampus among the four groups (P>0.05). Compared with group C, the expression of cleaved caspase-3 was significantly up-regulated, and the expression of p-ERK1/2 was down-regulated in K and G15EK groups (P<0.05). Compared with group K, the expression of cleaved caspase-3 was significantly down-regulated, and the expression of p-ERK1/2 was up-regulated in group EK (P<0.05). Compared with group EK, the expression of cleaved caspase-3 was significantly up-regulated, and the expression of p-ERK 1/2 was down-regulated in group G15EK (P<0.05).@*Conclusion@#GPR30 is involved in 17β estradiol-induced inhibition of ketamine-caused neuroapoptosis in the hippocampus of newborn rats, which is related to up-regulating the expression of p-ERKl/2.

Role of ERK signaling pathway in intrathecal dexmedetomidine-induced reduction of spinal cord ischemia-reperfusion injury in rats / 中华麻醉学杂志

Objective To evaluate the role of extracellular signal-regulated kinase (ERK) signaling pathway in intrathecal dexmedetomidine-induced reduction of spinal cord ischemia-reperfusion (I/R)injury in rats.Methods Eighty clean-grade male Sprague-Dawley rats,aged 9-10 weeks,weighing 300-350 g,were divided into 4 groups (n =20 each) using a random number table method:sham operation group (group S),spinal cord I/R group (group I/R),dexmedetomidine group (group D),and dexmedetomidine plus ERK signaling pathway blocker PD98059 group (group P).Spinal cord ischemia was produced by cross-clamping of the abdominal aorta distal to the left renal artery for 25 min followed by reperfusion to establish the model of spinal cord I/R injury.Dexmedetomidine 1 μg/kg was intrathecally injected at 20 min before establishing the model in D and P groups,PD98059 2 mg/kg was given via the tail vein at the same time in group P,and the equal volume of normal saline was given instead in S and I/R groups.Five rats were selected at 6,8,10 and 12 h of reperfusion,and the modified Basso,Beattie,Bresnahan (BBB) scale was used to assess the hindlimb locomotor function.Five rats were sacrificed after assessing the locomotor function at 6 h of reperfusion,and the L3-5 segments of the spinal cord were taken for determination of cell apoptosis (by TUNEL) and expression of phosphorylated ERK (p-ERK) (by Western blot).The apoptosis index was calculated.Results Compared with group S,the BBB scores were significantly decreased at each time point of reperfusion,the apoptosis index was increased,and the expression of pERK was up-regulated in the other three groups (P＜0.05).Compared with I/R group,the BBB scores were significantly increased at each time point of reperfusion,the apoptosis index was increased,and the expression of p-ERK was up-regulated in group D,and no significant change was found in the apoptosis index or p-ERK expression in group P (P＞0.05).Compared with group D,the BBB scores were significantly decreased at each time point of reperfusion,the apoptosis index was increased,and the expression of pERK was down-regulated in group P (P＜0.05).Conclusion The mechanism by which intrathecal dexmedetomidine reduces spinal cord I/R injury is related to activating ERK signaling pathway in rats.

Role of GPR30 in 17β estradiol-induced inhibition of ketamine-caused neuroapoptosis in hippocampus of newborn rats: the relationship with p-ERK1/2 / 中华麻醉学杂志

Objective To evaluate the role of G protein-coupled receptor 30 (GPR30) in 17β estradiol-induced inhibition of ketamine-caused neuroapoptosis in the hippocampus of newborn rats and the relationship with phosphorylated extracellular signal-regulated kinase 1/2 (p-ERKl/2).Methods Twentyfour clean-grade healthy male Sprague-Dawley rats,aged 7 days,weighing 11-18 g,were divided into 4 groups (n =6 each) using a random number table method:control group (group C),ketamine group (group K),17β estradiol plus ketamine group (group EK),and GPR30 inhibitor G15 plus 17β estradiol plus ketamine group (group G15EK).Ketamine 75 mg/kg (diluted to 0.1 ml in normal saline) was intraperitoneally injected every 24 h for 3 consecutive days in group K.In group EK,17β estradiol 600 μg/kg was subcutaneously injected and ketamine 75 mg/kg was intraperitoneally injected every 24 h for 3 consecutive days.G15 300 μg/kg and 17β estradiol 600 μg/kg were subcutaneously injected and ketamine 75 mg/kgwas intraperitoneally injected every 24 h for 3 consecutive days in group G15EK.The equal volume of normal saline 0.1 ml was intraperitoneally injected instead in group C.The animals were sacrificed at 24 h after the last injection for determination of the expression of cleaved caspase-3,ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) (by Western blot).Results There was no significant difference in the expression of ERK1/2 in hippocampus among the four groups (P＞0.05).Compared with group C,the expression of cleaved caspase-3 was significantly up-regulated,and the expression of p-ERK1/2 was down-regulated in K and G15EK groups (P＜0.05).Compared with group K,the expression of cleaved caspase-3 was significantly down-regulated,and the expression of p-ERK1/2 was up-regulated in group EK (P＜0.05).Compared with group EK,the expression of cleaved caspase-3 was significantly up-regulated,and the expression of p-ERK 1/2 was down-regulated in group G15EK (P＜0.05).Conclusion GPR30 is involved in 17β estradiol-induced inhibition of ketamine-caused neuroapoptosis in the hippocampus of newborn rats,which is related to up-regulating the expression of p-ERKl/2.

Role and mechanism of nonreceptor tyrosine kinase Tec in endotoxin/lipopolysaccharide-induced interleukin-8 production in human alveolar epithelial cells A549 / 中华烧伤杂志

Objective@#To investigate the role and mechanism of nonreceptor tyrosine kinase Tec in the production of pro-inflammatory cytokine interleukin-8 (IL-8) induced by endotoxin/lipopolysaccharide (LPS) in human alveolar epithelial cells A549.@*Methods@#Human alveolar epithelial cells A549 were routinely cultured and passaged in Roswell Park Memorial Institute-1640 medium containing 10% fetal bovine serum. The second or third passage of cells were collected for subsequent experiments. (1) Cells were collected and divided into 6 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in simple LPS group were routinely cultured for 1 h and then stimulated by 1 μg/mL LPS for 1 h. Cells in simple LFM-A13 group were cultured with conventional culture medium adding 75 μmol/L LFM-A13 for 1 h and then cultured with replaced conventional culture medium for 1 h. Cells in 25 μmol/L LFM-A13+ LPS group, 75 μmol/L LFM-A13+ LPS group, and 100 μmol/L LFM-A13+ LPS group were cultured with conventional culture medium adding 25, 75, and 100 μmol/L LFM-A13 respectively for 1 h and then all stimulated by 1 μg/mL LPS added into the replaced conventional culture medium for 1 h. The protein expression of Tec in cells of each group was detected by Western blotting, and the content of IL-8 in cell culture supernatant of each group was determined by enzyme-linked immunosorbent assay. (2) Cells were collected and divided into 5 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in small interfering RNA (siRNA) control+ LPS group were transfected with empty lentivirus for 10 h and then stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. Cells in Tec mus-298 RNA interference (RNAi)+ LPS group, Tec mus-299 RNAi+ LPS group, and Tec mus-300 RNAi+ LPS group were transfected with lentivirus loaded with Tec mus-298 RNAi, Tec mus-299 RNAi, and Tec mus-300 RNAi respectively for 10 h and then stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. The protein expression of Tec in cells of each group was detected by Western blotting to screen Tec-siRNA with the best silencing effect on Tec gene. (3) Cells were collected and divided into 4 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in virus control group were transfected with empty lentivirus for 10 h and then routinely cultured for 2 h. Cells in simple LPS group were stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. Cells in Tec-siRNA+ LPS group were transfected with lentivirus loaded with Tec-siRNA with the best silencing effect on Tec gene for 10 h and then stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. The protein expressions of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) MAPK of cells in each group were detected by Western blotting. Data were processed with one-way analysis of variance and the least significant difference-t test.@*Results@#(1) Compared with that of blank control group, the protein expression of Tec of cells in simple LPS group was obviously increased (t=9.72, P<0.05), but the protein expression of Tec of cells in simple LFM-A13 group was not obviously changed (t=4.31, P=0.05). Compared with that of simple LPS group, the protein expression of Tec of cells in 25 μmol/L LFM-A13+ LPS group, 75 μmol/L LFM-A13+ LPS group, or 100 μmol/L LFM-A13+ LPS group was obviously decreased (t=9.72, 9.07, 16.33, P<0.05 or P<0.01). Compared with (189±22) pg/mL of blank control group, the content of IL-8 in culture supernatant of cells in simple LPS group was obviously increased [(214±10) pg/mL, t=2.18, P<0.05], but the content of IL-8 in culture supernatant of cells in simple LFM-A13 group was not obviously changed [(173±43) pg/mL, t=0.64, P>0.05]. Compared with that of simple LPS group, the content of IL-8 in culture supernatant of cells in 25 μmol/L LFM-A13+ LPS group was not obviously changed [(204±38) pg/mL, t=0.54, P>0.05], but the content of IL-8 in culture supernatant of cells in 75 μmol/L LFM-A13+ LPS group and 100 μmol/L LFM-A13+ LPS group was obviously decreased [(144±44), (137±51) pg/mL, t=3.63, 2.55, P<0.05 or P<0.01]. (2) Compared with that of blank control group, the protein expression of Tec of cells in siRNA control+ LPS group was obviously increased (t=14.24, P<0.01). Compared with that of siRNA control+ LPS group, the protein expression of Tec of cells in Tec mus-298 RNAi+ LPS group or Tec mus-299 RNAi+ LPS group was obviously decreased (t=36.03, 18.23, P<0.01), but the protein expression of Tec of cells in Tec mus-300 RNAi+ LPS group was not obviously changed (t=4.08, P>0.05). The protein expression of Tec was the lowest in cells of Tec mus-298 RNAi+ LPS group, so Tec mus-298 RNAi was used in subsequent experiment. (3) Compared with 1.16±0.16 and 0.78±0.11 of blank control group, the protein expressions of p38 MAPK and ERK MAPK of cells in virus control group were not obviously changed (1.66±0.13, 0.89±0.11, t=11.09, 3.60, P>0.05), but the protein expressions of p38 MAPK and ERK MAPK of cells in simple LPS group were obviously increased (2.83±0.29, 1.86±0.37, t=9.70, 7.23, P<0.05). Compared with those of simple LPS group, the protein expression of p38 MAPK and protein expression of ERK MAPK of cells in Tec-siRNA+ LPS group were obviously decreased (0.69±0.16, 1.03±0.24, t=13.78, 4.12, P<0.05 or P<0.01).@*Conclusions@#Tec may mediate the production and release of pro-inflammatory cytokine IL-8 from human alveolar epithelial cells A549 induced by LPS via the p38 MAPK and ERK MAPK signal pathways.

The effect of DUSP2 on proliferation and apoptosis of gastric cancer cells and its mechanism / 天津医药

@#Objective To investigate the effect of dual-specificity phosphatase-2 (DUSP2) on the cell proliferation and apoptosis in the gastric cancer and its mechanisms. Methods Firstly, the effects of different expressions of DUSP2 on the overall survival of 876 gastric cancer patients were analyzed by online analysis tool KM plotter, and the expressions of DUSP2 in various gastric cancer cell lines (MKN-45, SGC-7901, HGC-27 and N-87) were verified. Secondly, DUSP2 overexpressed lentiviral vector was constructed, and MKN-45 was transfected by packaged virus. DUSP2-overexpression gastric cancer cell line was gained by drug screening. Meanwhile, gastric cancer cells infected with empty vector virus were used as control. Then the effect of DUSP2 upregulation on the proliferation ability of gastric cancer cells was evaluated by MTS cell proliferation assay, and the apoptosis was determined by Annexin V-FITC / PI double staining. The protein expressions of DUSP2, ERK, p-ERK (Thr202/Tyr204), P38 and p-P38 were tested by the Western blot analysis. Results Gastric cancer patients with high DUSP2 expression showed a significant survival advantage compared with those with low DUSP2 expression, and DUSP2 levels were decreased in several gastric cancer cell lines. The Western blot analysis revealed that the expression of DUSP2 markedly increased in overexpressed DUSP2 group (experimental group) compared with that of control group. The MTS experiment showed that the cell viability was significantly decreased in experimental group than that of the control group. Correspondingly, the cell apoptosis test showed that the cell apoptosis rate was obviously higher in the experimental group than that of the control group. The results of Western blot assay indicated that p-ERK (Thr202/Tyr204) and p-38 were significantly down-regulated in the experimental group compared with those of control group. Conclusion The over-expression of DUSP2 can efficiently inhibit cell proliferation and promote its apoptosis in gastric cancer cells, and the mechanism is related to DUSP2 inhibiting the phosphorylation levels of ERK and P38.

Protective effect of WSY6, a caffeic acid derivative, on oxidative stress injury in melanocytes and its mechanism / 中华皮肤科杂志

Objective To evaluate the protective effect of WSY6 (a caffeic acid derivative) on hydrogen peroxide (H2O2)-induced oxidative stress injury in melanocytes,and to explore its potential molecular mechanism.Methods In vitro cultured human primary melanocytes were divided into 5 groups:control group receiving no treatment,H2O2 group treated with 1 mmol/L H2O2,6.25,12.5,25 μmol/L WSY6 groups pretreated with 6.25,12.5,25 μmol/L WSY6 respectively followed by 1-hour treatment with 1 mmol/L H2O2.After 24-hour treatment,MTS assay was performed to determine the survival rate of melanocytes,and the lactate dehydrogenase (LDH)kit was used to detect the LDH leakage level.Some melanocytes were divided into 2 groups:inhibitor group pretreated with the p38 inhibitor for 1 hour followed by 1-hour treatment with 1 mmol/L H2O2,and H2O2 group treated with 1 mmol/L H2O2 for 1 hour.After 24-hour treatment,the LDH kit was used to detect the LDH leakage level.Some other melanocytes were pretreated with 25 μmol/L WSY6 for 1,2,4 hours separately,followed by 1-hour treatment with H2O2.Then,flow cytometry was conducted to detect the level of intracellular reactive oxygen species (ROS).Some melanocytes were treated with 6.25,12.5,25 μmol/L WSY6 separately for 1 hour,followed by 1-hour treatment with H2O2.Then,Western bolt analysis was performed to determine the protein expression of cytochrome c (cyto-c),caspase-3,caspase-9,phosphorylated (p)-p38 MAPK,p-ERK and p-JNK.Results Compared with the control group,the H2O2 group showed significantly decreased survival rate of melanocytes (29.22％ ± 1.31％,P ＜ 0.05),but significantly increased intracellular LDH leakage level (47.19％ ± 4.85％,P ＜ 0.05),elevated intracellular ROS level (18.37 ± 1.59,P ＜ 0.05),and increased expression of caspase-3 and caspase-9.Compared with the H2O2 group,the 6.25,12.5,25 μmol/L WSY6 groups showed significantly increased cell survival rate (52.48％ ± 1.17％,60.21％ ± 0.25％,78.32％ ± 1.73％,P ＜ 0.05),but significantly decreased LDH leakage level (21.99％ ± 0.22％,15.38％ ± 0.45％,13.18％ ± 0.38％,P ＜ 0.05),and the intracellular ROS level was significantly decreased in the 25 μmol/L WSY6 group after 1,2,4 hours of treatment (7.59 ± 1.00,6.22 ± 0.52,5.1 ± 0.48,P ＜ 0.05).The LDH leakage level in melanocytes was significantly lower in the p38 inhibitor group than in the H2O2 group (P ＜ 0.05).Western blot analysis revealed that after the pretreatment with 6.25,12.5,25 μmol/L WSY6 separately,the WSY6 groups all showed obviously decreased expression of caspase-3,caspase-9 and p-p38 compared with the H2O2 group.However,there was no obvious difference in the expression of p-ERK and p-JNK between the WSY6 groups and the H2O2 group.Besides,the WSY6 groups showed decreased expression of p-p53 (a downstream product of p38 MAPK),which decreased along with the increase in the concentration of WSY6.Conclusion WSY6 shows a markedly protective effect on H2O2-induced oxidative stress injury in melanocytes,likely through the p38 MAPK pathway.

Relationship between SIRT1-ERK1∕2 pathway and chikusetsu saponin IVa-induced reduction of isoflurane-elicited neurotoxicity in fetal rats: an in vitro experiment / 中华麻醉学杂志

Objective To evaluate the relationship between silent information regulator 1 ( SIRT1)-extracellular-regulated kinase1∕2 ( ERK1∕2) pathway and chikusetsu saponin IVa-induced reduc-tion of isoflurane-elicited neurotoxicity in fetal rats in an in vitro experiment. Methods The hippocampal neurons isolated from rats at 16-18 days of gestation were primarily cultured for 7 days and divided into 3 groups ( n = 6 each) using a random number table method: control group ( Con group) , isoflurane group (Iso group) and chikusetsu saponin IVa plus isoflurane group (ChIV+Iso group). Hippocampal neurons were cultured routinely for 6 h in Con group. Hippocampal neurons were exposed to 1. 8％ isoflurane for 6 h in an incubator in Iso group. Chikusetsu saponin IVa 25μg∕ml was added to the culture medium, and hipp-ocampal neurons were incubated for 6 h and then exposed to 1. 8％ isoflurane for 6 h in an incubator in ChIV+Iso group. The supernatant was collected for determination of the amount of lactic dehydrogenase ( LDH) released, neuronal viability ( by CCK-8) and expression of SIRT1, ERK1∕2 and phosphorylated ERK1∕2 ( p-ERK1∕2) ( by Western blot) . Results Compared with Con group, the neuronal viability was significantly decreased, the amount of LDH released was increased, and the expression of SIRT1 and p-ERK1∕2 was down-regulated in Iso group ( P<0. 05) . Compared with Iso group, the neuronal viability was significantly increased, the amount of LDH released was decreased, and the expression of SIRT1 and p-ERK1∕2 was up-regulated in ChIV+Iso group ( P<0. 05) . Conclusion The mechanism by which chikuset-su saponin IVa reduces isoflurane-elicited neurotoxicity is related to activating SIRT1-ERK1∕2 pathway in fe-tal rats in an in vitro experiment.

Role of hippocampal ERK1/2 in exogenous carbon monoxide-induced improvement of cognitive function in a rat model of hemorrhage shock and resuscitation / 中华麻醉学杂志

Objective To evaluate the role of hippocampal extracellular signal-regulated kinase 1/2 (ERK1/2) in exogenous carbon monoxide (CO)-induced improvement of cognitive function in a rat model of hemorrhage shock and resuscitation.Methods Ninety clean-grade healthy male Sprague-Dawley rats,aged 9-10 weeks,weighing 350-400 g,were divided into 5 groups (n=18 each) using a random number table method:sham operation group (S group),hemorrhage shock and resuscitation group (H group),CO group,PD98059-CO group (PCO group) and PD98059 group (P group).Hemorrhagic shock was induced by withdrawing blood from the femoral vein until mean arterial pressure was reduced to 25-35 mmHg which was maintained for 60 min and resuscitated by infusing the blood withdrawn over 15 min until the initial blood pressure was achieved,and normal saline was infused when needed.Rats were exposed to air mixture containing 1％ CO for 3 h in a glass box after the end of resuscitation in group CO.ERK1/2 inhibitor PD98059 (30 μmol/L) 30 μl was injected into the cerebral ventricle at 30 min before hemorrhage in PCO and PC groups.Right femoral artery and vein were only cannulated,and normal saline was injected into the cerebral ventricle in group S.Rats were sacrificed at 3 h after the end of resuscitation,brains were removed and hippocampi were isolated for determination of CO content (by gas chromatograph assay).Cognitive function was assessed by Morris water maze test at 15 days after the end of resuscitation,rats were then sacrificed and hippocampi were isolated for determination of cell apoptosis in hippocampal CA1 region by TUNEL and cleaved caspase-3 immunofluorescence,and the apoptosis rate was calculated.Rats were sacrificed at 6 h after the end of resuscitation,and hippocampi were isolated to detect the expression of phosphorylated ERK1/2 (p-ERK1/2),Bcl-2 and Bax by Western blot.Results Compared with group S,the escape latency was significantly prolonged in H,PCO and P groups,and the hippocampal CO content and apoptosis rate were increased,the expression of cleaved caspase-3 and ERK1/2 was up-regulated,and the Bcl-2/Bax ratio was decreased in H,CO,PCO and P groups (P＜0.05).Compared with group H,the escape latency was significantly shortened,the hippocampal CO content was increased,and the apoptosis rate was decreased,the cleaved caspase-3 expression was down-regulated,p-ERK1/2 expression was upregulated,and Bcl-2/Bax ratio was increased in group CO,and the escape latency was significantly prolonged,the hippocampal CO content was decreased,the apoptosis rate was increased,the cleaved caspase-3 expression was up-regulated,p-ERK1/2 expression was down-regulated,and Bcl-2/Bax ratio was decreased in group P (P＜0.05).Compared with group CO,the escape latency was significantly prolonged,the apoptosis rate was increased,the cleaved caspase-3 expression was up-regulated,p-ERK1/2 expression was down-regulated,and Bcl-2/Bax ratio was decreased in group PCO (P＜0.05).Conclusion The mechanism by which exogenous CO improves cognitive function is related to raising the phosphorylation of ERK1/2 in hippocampal neurons and inhibiting neuronal apoptosis in a rat model of hemorrhage shock and resuscitation.

Effect of proliferation of triptolide on MV4-11 cells and its mechanism research associated with RAS/ERK/MAPK / 中国医师杂志

Objective To explore the effect of proliferation and apoptosis,and research its mechanism associated with RAS/extracellular signal regulated kinase/mitogen-activated protein kinase (RAS/ ERK/MAPK) in Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutation acute lymphocytic leukemia cell line MV4-11 cells treated by triptolide (TP).Methods MV4-11 cells were respectively treated by triptolide with diverse concentrations and different times.The proliferation inhibition rate was measured by methyl thiazolyl tetrazolium,the apoptosis rate was detected by flow cytometry,the mRNA expressions of FLT3,RAS,ERK,forkhead box protein M1 (FOXM1),and c-Myc were analyzed by realtime fluorescent quantitative polymerase chain reaction (PCR).Results The proliferation inhibition rate markedly increased in a dose-time dependent manner after MV4-11 cells were treated by TP.After cells were treated with 10,and 20 nmol/L TP,respectively,the apoptosis rates at 48 h were (17.30 ± 0.56) ％,(35.77 ±0.55)％,and those at 72 h were (49.83 ±0.45)％,(68.90 ±0.75)％ correspondingly.PCR data showed that the mRNA level of FLT3 mRNA decreased obviously,and that of RAS,ERK,FOXM1,and c-Myc also decreased.Conclusions Triptolide could significantly inhibit the MV4-11 cells proliferation and induce cell apoptosis,and its mechanism might be through inhibiting the expression of related genes on RAS/ERK/MAPK signaling pathway.

Role of M3 receptor in penehyclidine hydrochloride-induced reduction of increased permeability of human pulmonary microvascular endothelial cells caused by endotoxin: the relationship with MAPK signaling pathway / 中华麻醉学杂志

Objective To evaluate the role of M3 receptor in penehyclidine hydrochloride(PHC)-induced reduction of increased permeability of human pulmonary microvascular endothelial cells (PMVECs) caused by endotoxin and the relationship with mitogen-activated protein kinase (MAPK) signaling pathway.Methods Human PMVECs were seeded in 6-well plates (2 ml/hole) or in culture flasks (4 ml/flask) at the density of 1 × 105 cells/ml and randomly divided into 6 groups (n=5 each):control group (group C),M3 receptor shRNA transfection group (group shRNA),lipopolysaccharide (LPS) group,penehyclidine plus LPS group (group P+LPS),LPS plus M3 receptor shRNA transfection group (group LPS+shRNA) and PHC plus LPS plus M3 shRNA transfection group (group P+LPS+shRNA).The cells were transfected with shRNA plasmid containing 2.5 nmol/L M3 receptors in shRNA,LPS+shRNA and P+LPS+shRNA groups.LPS at the final concentration of 0.1 μg/ml was added at 24 h of incubation and then cells were incubated for 1 h in LPS and LPS+shRNA groups.PHC at the final concentration of 2 μg/ml was added at 24 h of incubation,cells were incubated for 1 h,then LPS at the final concentration of 0.1 μg/ml was added,and cells were incubated for another l h in P+LPS and P+LPS+shRNA groups.The permeability of PMVECs was measured using Transwell assay.The expression of phosphorylated p38 MAPK (p-p38 MAPK)and phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) was detected by Western blot,the expression of heat shock protein 27 (HSP27) using immunofluorescent staining,and the expression of M3receptor mRNA by real-time polymerase chain reaction.Results Compared with group C,M3 receptor mRNA expression was significantly down-regulated in group shRNA,and the permeability of cells was significantly increased,and the expression of p-p38 MAPK,p-ERK1/2,HSP27 and M3 receptor mRNA was up-regulated in group LPS (P＜0.05).The permeability of cells was significantly decreased,and the expression of p-p38 MAPK,p-ERK1/2,HSP27 and M3 receptor mRNA was down-regulated in P+ LPS,LPS+shRNA and P+LPS+shRNA groups as compared with group LPS,and in group P+LPS+shRNA as compared with group LPS+shRNA (P＜0.05).Conclusion The mechanism by which PHC reduces endotoxin-caused increased permeability of human PMVECs is related to inhibiting activation of MAPK signaling pathway after down-regulating M3 receptor.

Various Wavelengths of Light-Emitting Diode Light Regulate the Proliferation of Human Dermal Papilla Cells and Hair Follicles via Wnt/β-Catenin and the Extracellular Signal-Regulated Kinase Pathways

BACKGROUND: The human dermal papilla cells (hDPCs) play an important role in regulation of hair cycling and growth. OBJECTIVE: The aim of this study was to investigate the effect of different wavelengths of light-emitting diode (LED) irradiation on the proliferation of cultured hDPCs and on the growth of human hair follicles (HFs) in vitro. METHODS: We examined the effect of LED irradiation on Wnt/β-catenin signaling and mitogen-activated protein kinase (MAPK) pathways in hDPCs. Anagen HFs were cultured with LED irradiation and elongation of each hair shaft was measured. RESULTS: The most potent wavelength in promoting the hDPC proliferation is 660 nm and 830 nm promoted hDPC proliferation to a lesser extent than 660 nm. Various wavelengths significantly increased β-catenin, Axin2, Wnt3a, Wnt5a and Wnt10b mRNA expression. LED irradiation significantly increased β-catenin and cyclin D expression, and the phosphorylation of MAPK and extracellular signal-regulated kinase (ERK). HFs irradiated with 415 nm and 660 nm grew longer than control. CONCLUSION: Our result suggests that LED has a potential to stimulate hDPC proliferation via the activation of Wnt/β-catenin signaling and ERK pathway. To our best knowledge, this is the first report which investigated that the effect of various wavelengths of LED on hDPC proliferation and the underlying mechanisms.

Testosterone and Voluntary Exercise, Alone or Together Increase Cardiac Activation of AKT and ERK1/2 in Diabetic Rats / Testosterona e Exercício Voluntário, Individualmente ou em Conjunto, Aumentam a Ativação Cardíaca de AKT e ERK1/2 em Ratos Diabéticos

Abstract Background: Impaired angiogenesis in cardiac tissue is a major complication of diabetes. Protein kinase B (AKT) and extracellular signal regulated kinase (ERK) signaling pathways play important role during capillary-like network formation in angiogenesis process. Objectives: To determine the effects of testosterone and voluntary exercise on levels of vascularity, phosphorylated Akt (P- AKT) and phosphorylated ERK (P-ERK) in heart tissue of diabetic and castrated diabetic rats. Methods: Type I diabetes was induced by i.p injection of 50 mg/kg of streptozotocin in animals. After 42 days of treatment with testosterone (2mg/kg/day) or voluntary exercise alone or in combination, heart tissue samples were collected and used for histological evaluation and determination of P-AKT and P-ERK levels by ELISA method. Results: Our results showed that either testosterone or exercise increased capillarity, P-AKT, and P-ERK levels in the heart of diabetic rats. Treatment of diabetic rats with testosterone and exercise had a synergistic effect on capillarity, P-AKT, and P-ERK levels in heart. Furthermore, in the castrated diabetes group, capillarity, P-AKT, and P-ERK levels significantly decreased in the heart, whereas either testosterone treatment or exercise training reversed these effects. Also, simultaneous treatment of castrated diabetic rats with testosterone and exercise had an additive effect on P-AKT and P-ERK levels. Conclusion: Our findings suggest that testosterone and exercise alone or together can increase angiogenesis in the heart of diabetic and castrated diabetic rats. The proangiogenesis effects of testosterone and exercise are associated with the enhanced activation of AKT and ERK1/2 in heart tissue.

Resumo Fundamento: Angiogênese prejudicada em tecido cardíaco é uma das principais complicações das diabetes. As vias de sinalização da proteína-quinase B (AKT) e a quinase regulada por sinal extracelular (ERK) exercem um importante papel durante a formação de uma rede similar à capilar no processo de angiogênese. Objetivos: Determinar os efeitos da testosterona e exercícios voluntários sobre os níveis de vascularidade, AKT fosforilada (P- AKT) e ERK fosforilada (P-ERK) sobre o tecido cardíaco de ratos diabéticos e castrados diabéticos. Métodos: A diabetes tipo 1 foi induzida através de injeção intraperitoneal de 50 mg/kg de estreptozotocina em animais. Após 42 dias de tratamento com testosterona (2mg/kg/dia) ou exercícios voluntários, individualmente ou em conjunto, as amostras de tecidos cardíacos foram coletadas e usadas para avaliação histológica e determinação de níveis de P-AKT e P-ERK através do método ELISA. Resultados: Os nossos resultados mostraram que a testosterona ou os exercícios aumentaram a capilaridade, os níveis de P-AKT, e P-ERK nos corações de ratos diabéticos. O tratamento de ratos diabéticos com testosterona e exercícios obteve um efeito sinérgico sobre a capilaridade, níveis de P-AKT, e P-ERK no coração. Além disto, na capilaridade do grupo diabético castrado, os níveis de P-AKT e P-ERK diminuíram significativamente no coração, ao passo que o tratamento com testosterona ou o treinamento com exercícios reverteu tais efeitos. O tratamento simultâneo de ratos diabéticos castrados com testosterona e exercícios obteve um efeito aditivo sobre os níveis de P-AKT e P-ERK. Conclusão: Nossas descobertas sugerem que a testosterona e exercícios, em conjunto ou individualmente, podem aumentar a angiogênese nos corações de ratos diabéticos e castrados diabéticos. Os efeitos favoráveis à angiogênese da testosterona e dos exercícios estão associados à ativação reforçada de AKT e ERK1/2 no tecido cardíaco.

Influence of extracellular signal-regulated kinase 1/2 pathway inhibitor PD98059 on proliferation and killing function of γδT cells cultured in vitro / 临床肝胆病杂志

ObjectiveTo investigate the effect of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway inhibitor PD98059 on the proliferation and killing function of γδT cells cultured in vitro. MethodsMononuclear cells were separated from peripheral blood in healthy subjects and placed in RPMI 1640 complete medium containing zoledronic acid and interleukin-2 to obtain γδT cells through induction and culture. The ERK1/2 specific inhibitor PD98059 was used to block the ERK1/2 signaling pathway in γδT cells, and the proliferation of γδT cells was measured by cell counting, and the killing function of γδT cells was measured by CCK-8 method. Flow cytometry was used to measure the expression of granzyme B and perforin in γδT cells. The t-test was used for comparison of continuous data between groups. ResultsAfter 10 days of culture, the purity of γδT cells reached 87.94%±2.36%. The number of γδT cells treated with PD98059 was significantly lower than that of control cells ［(6.74±0.36)×105/ml vs (9.42±0.31)×105/ml, t=-12.708, P＜0.001］. Compared with the control cells, those treated with PD98059 had significantly higher positive expression rates of granzyme B and perforin (granzyme B: 48.89%±1.31% vs 41.58%±1.58%, t=7.582, P＜0.001; perforin: 65.92%±3.29% vs 33.49%±2.83%, t=15.478, P＜0001) and a significantly higher killing rate of HepG2 cells (69.28%±4.96% vs 48.34%±3.01%, t=11.201, P＜0.001). ConclusionThe ERK1/2 specific inhibitor PD98059 can inhibit the proliferation of γδT cells, but it can enhance the in vitro killing function of γδT cells.

Role of β-arrestin-1 in inhibition of endoxin-induced activation of MAPK signaling pathway in pulmonary microvascular endothelial cells by penehyclidine hydrochloride / 中华麻醉学杂志

Objective To investigate the role of β-arrestin-1 in inhibition of endoxin-induced activation of MAPK signaling pathway in pulmonary microvascular endothelial cells (PMVECs) by penehyclidine hydrochloride (PHC).Methods Human PMVECs were seeded in 6-well plates (2 ml/well) or in culture flasks (4 ml/flask) at the density of 1×105 cells/ml,and randomly divided into 5 groups (n=20 each) using a random number table:empty plasmid transfection group (group C),lipopolysaccharide (LPS) + empty plasmid transfection group (LPS group),PHC + LPS + empty plasmid transfection group (P + LPS group),LPS+β-arrestin-1 short hairpin RNA (shRNA) transfection group (LPS+shRNA group),and PHC + LPS+β-arrestin-1 shRNA transfection group (P+LPS+shRNA group).After the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA,the cells were incubated for 24 h.At 24 h of incubation,LPS with the final concentration of 0.1 μg/ml was added,and the cells were then incubated for 1 h in LPS and LPS+ shRNA groups.In P+LPS and P+LPS+shRNA groups,PHC with the final concentration of 2 μg/ml was added,and the cells were incubated for 1 h,and then LPS with the final concentration of 0.1 μg/ml was added,and the cells were incubated for 1 h.The expression of filamentous actin (F-actin) was detected by flow cytometry.The expression of myosin light chain kinase (MLCK) and vascular endothelial-cadherin (VE-cadherin) was detected by immunofluorescence.The expression of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phosphorylated cJun N-terminal kinase (p-JNK) was determined by Western blot.The expression of β-arrestin-1 mRNA was determined by real-time polymerase chain reaction.Results Compared with group C,the expression of Factin,VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated,and the expression of MLCK,p-ERK1/2 and p-JNK was up-regulated in group LPS,and the expression of p-ERK1/2 and p-JNK was significantly up-regulated (P＜0.05),and no significant change was found in the other parameters mentioned above in group P+LPS (P＞0.05).Compared with group LPS,the expression of F-actin,VE-cadherin and β-arrestin-1 mRNA was significantly up-regulated,and the expression of MLCK,p-ERK1/2 and p-JNK was down-regulated in group P+LPS,and the expression of F-actin,VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated,and the expression of MLCK and p-JNK was up-regulated in group LPS+shRNA (P＜0.05).Compared with group P+LPS,the expression of F-actin,VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated,and the expression of MLCK,p-ERK1/2 and p-JNK was up-regulated in group P+LPS+shRNA (P＜0.05).Conclusion The mechanism by which PHC inhibits endoxin-induced activation of MAPK signaling pathway in PMVECs is partially related to up-regulation of β-arrestin-1 expression.