小白菊内酯调节JAK/STAT信号通路对高糖诱导胰岛β细胞损伤的保护作用

Protective effect of parthenolide on high glucose induced isletβcell injury via regulating JAK/STAT signaling pathway

目的 探讨小白菊内酯(PTL)对高糖(HG)诱导胰岛β细胞损伤的影响及机制。方法 分别用0、2.5、5、10、20、40μmol/L PTL处理HG诱导的小鼠胰岛β细胞MIN6细胞,通过检测细胞活力筛选出合适的处理MIN6细胞的PTL浓度;取对数生长期的MIN6细胞,分为对照组(5.5 mmol/L葡萄糖)、HG组(33.3 mmol/L葡萄糖)、HG+低剂量PTL组(33.3 mmol/L葡萄糖+2.5μmol/L PTL)、HG+中剂量PTL组(33.3 mmol/L葡萄糖+5μmol/L PTL)、HG+高剂量PTL组(33.3 mmol/L葡萄糖+10μmol/L PTL)、HG+AG490[Janus激酶(JAK)/(信号转导和转录激活子STAT)通路抑制剂]组(33.3mmol/L葡萄糖+40μmol/L AG490)、HG+高剂量PTL+AG490组(33.3 mmol/L葡萄糖+10μmol/L PTL+40μmol/L AG490)。CCK-8检测MIN6细胞增殖;流式细胞术检测MIN6细胞凋亡;酶联免疫吸附试验检测细胞上清液中白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平及胰岛素含量;Western blot检测MIN6细胞中增殖细胞核抗原(PCNA)、Bcl-2相关X蛋白(Bax)、磷酸化Janus激酶2(p-JAK2)、磷酸化信号转导和转录激活子3(p-STAT3)蛋白表达。结果 与对照组比较,HG组OD450值[(0.23±0.02)比(1.14±0.12),P<0.05]、胰岛素含量[(224.46±9.83)U/mL比(368.54±14.45)U/mL,P<0.05]、PCNA[(0.39±0.03)比(1.64±0.14),P<0.05]、p-JAK2[(0.25±0.02)比(0.92±0.08),P<0.05]、pSTAT3 [(0.29±0.02)比(0.83±0.08),P <0.05]蛋白表达降低,细胞凋亡率[(16.67±0.74)%比(3.54±0.16)%,P<0.05]、IL-6、TNF-α水平、Bax[(1.24±0.11)比(0.28±0.02),P<0.05]蛋白表达升高。与HG组比较,HG+低剂量PTL组、HG+中剂量PTL组、HG+高剂量PTL组OD450值[(0.36±0.03)、(0.58±0.05)、(0.86±0.08)比(0.23±0.02),P <0.05]、胰岛素含量、PCNA [(0.67±0.05)、(0.94±0.09)、(1.37±0.13)比(0.39±0.03),P <0.05]、p-JAK2 [(0.36±0.03)、(0.52±0.05)、(0.78±0.06)比(0.25±0.02),P <0.05]、p-STAT3[(0.41±0.04)、(0.59±0.05)、(0.74±0.06)比(0.29±0.02),P<0.05]蛋白表达升高,细胞凋亡率[(14.45±0.61)%、(10.36±0.52)%、(5.84±0.21)%比(16.67±0.74)%,P<0.05]、IL-6、TNF-α水平、Bax[(0.96±0.08)、(0.63±0.05)、(0.44±0.03)比(1.24±0.11),P<0.05]蛋白表达降低。与HG组比较,HG+AG490组OD450值[(0.11±0.01)比(0.23±0.02),P<0.05]、胰岛素含量、PCNA [(0.15±0.01)比(0.39±0.03),P<0.05]、p-JAK2[(0.13±0.01)比(0.25±0.02),P<0.05]、p-STAT3[(0.16±0.01)比(0.29±0.02),P<0.05]蛋白表达降低,细胞凋亡率[(24.56±1.18)%比(16.67±0.74)%,P<0.05]、IL-6、TNF-α水平、Bax[(1.56±0.14)比(1.24±0.11),P<0.05]蛋白表达升高。AG490减弱了高剂量PTL对HG诱导的MIN6细胞损伤的改善作用。结论 PTL可能通过激活JAK/STAT信号通路促进HG诱导的MIN6细胞增殖,抑制细胞凋亡。

Objective To investigate the effect of parthenolide(PTL)on high glucose(HG)-induced injury in isletβ-cells and its underlying mechanism.Methods MIN6 cells,a mouse pancreaticβ-cell line,were treated with various concentrations of PTL(0,2.5,5,10,20,or 40μmol/L)under HG condition to determine the optimal concentration based on cell viability.MIN6 cells in the logarithmic growth phase were divided into the following groups:control group(5.5 mmol/L glucose),HG group(33.3 mmol/L glucose),HG+low-dose PTL group(33.3 mmol/L glucose+2.5μmol/L PTL),HG+medium-dose PTL group(33.3 mmol/L glucose+5μmol/L PTL),HG+high-dose PTL group(33.3 mmol/L glucose+10μmol/L PTL),HG+AG490[a Janus kinase/signal transducer and activator of transcription(JAK/STAT)pathway inhibitor]group(33.3 mmol/L glucose+40μmol/L AG490),and HG+high-dose PTL+AG490 group(33.3 mmol/L glucose+10μmol/L PTL+40μmol/L AG490).Cell proliferation was detected by CCK-8;the apoptosis of MIN6 cells was detected by flow cytometry;the levels of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and insulin in the cell supernatant were detected by enzyme-linked immunosorbent assay;and the expression of proliferating cell nuclear antigen(PCNA),BCL2 associated X protein(Bax),phosphorylated Janus kinase 2(p-JAK2),and phosphorylated signal transducer and activator of transcription 3(p-STAT3)proteins in MIN6 cells was detected by western blot.Results Compared to the control group,the HG group showed significantly decreased OD450 value[(0.23±0.02)vs.(1.14±0.12),P<0.05],insulin levels,PCNA expression[(0.39±0.03)vs.(1.64±0.14),P<0.05],p-JAK2 expression[(0.25±0.02)vs.(0.92±0.08),P<0.05],and p-STAT3 expression[(0.29±0.02)vs.(0.83±0.08),P<0.05],while the apoptosis rate[(16.67±0.74)%vs.(3.54±0.16)%,P<0.05],IL-6 levels,TNF-αlevels,and Bax expression[(1.24±0.11)vs.(0.28±0.02),P<0.05]were increased.Compared to the HG group,the HG+low-dose PTL,HG+medium-dose PTL,and HG+high-dose PTL groups showed increased OD450 value[(0.36±0.03),(0.58±0.05),(0.86±0.08)vs.(0.23±0.02),P<0.05],insulin levels,PCNA expression[(0.67±0.05),(0.94±0.09),(1.37±0.13)vs.(0.39±0.03),P<0.05],P-JAK2 expression[(0.36±0.03),(0.52±0.05),(0.78±0.06)vs.(0.25±0.02),P<0.05],and p-STAT3 expression[(0.41±0.04),(0.59±0.05),(0.74±0.06)vs.(0.29±0.02),P<0.05],while the apoptosis rate[(14.45±0.61)%,(10.36±0.52)%,(5.84±0.21)%vs.(16.67±0.74)%,P<0.05],IL-6 levels,TNF-αlevels,and Bax expression[(0.96±0.08),(0.63±0.05),(0.44±0.03)vs.(1.24±0.11),P<0.05]were decreased.Compared to the HG group,the HG+AG490 group showed decreased OD450 value[(0.11±0.01)vs.(0.23±0.02),P<0.05],insulin levels,PCNA expression[(0.15±0.01)vs.(0.39±0.03),P<0.05],p-JAK2 expression[(0.13±0.01)vs(0.25±0.02),P<0.05],and p-STAT3 expression[(0.16±0.01)vs(0.29±0.02),P<0.05],while the apoptosis rate[(24.56±1.18)%vs(16.67±0.74)%,P<0.05],IL-6 levels,TNF-αlevels,and Bax expression[(1.56±0.14)vs(1.24±0.11),P<0.05]were increased.AG490 attenuated the protective effect of high dose PTL on HG-induced injury of MIN6 cells.Conclusion PTL may promote proliferation and inhibit apoptosis of MIN6 cells under HG conditions by activating the JAK/STAT signaling pathway.