逍遥散含药血清影响RIN-m5f细胞活性及胰岛素分泌的机制探讨
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摘要
目的研究逍遥散含药血清对应激损伤状态糖尿病细胞模型[大鼠胰岛β细胞瘤细胞(RIN-m5f)]的活性、胰岛素(INS)分泌的影响及其干预机制。方法用高浓度葡萄糖(50 mmol·L-1)和皮质酮(CORT,320μmol·L-1)诱导培养RIN-m5f细胞12 h,制备应激损伤状态糖尿病细胞模型,再用30%逍遥散含药血清干预,并设立空白血清对照。共分为7组:①正常组:RIPM1640+10%FBS;②高糖组(G组):①+50 mmol·L-1的葡萄糖;③应激组(GCo组):②+320μmol·L-1的CORT;④空白血清高糖组(GC组):②+30%空白组大鼠血清(30%C)⑤逍遥散血清高糖组(GX组):②+30%逍遥散组大鼠血清(30%X);⑥空白血清应激组(GCoC组):③+30%C;⑦逍遥散血清应激组(GCoX组):③+30%X。干预12 h后,采用CCK-8法检测细胞活性;酶联免疫法(ELISA)检测细胞INS分泌水平;实时荧光定量PCR(qPCR)法检测细胞活化转录因子6(ATF-6)、肌醇需酶1(IRE-1)mRNA表达;Western Blot法检测细胞Caspase-12、CHOP蛋白表达。结果与正常组比较,G组细胞活性和ATF6、IRE1 mRNA以及Caspase-12、CHOP蛋白表达水平显著下降(P <0.05,P <0.01),INS水平显著升高(P <0.01)。与G组比较,GCo组细胞活性显著降低(P <0.01),INS水平和ATF6、IRE1 mRNA以及Caspase-12、CHOP蛋白表达水平均明显上调(P <0.05,P <0.01)。与GC组比较,GX组的细胞活性明显升高(P <0.05),ATF6、IRE1 mRNA以及Caspase-12蛋白表达水平均明显上调(P <0.05,P <0.01),但INS水平、CHOP蛋白表达水平差异无统计学意义(P>0.05)。与GCoC组比较,GCoX组的细胞活性、INS水平明显升高(P <0.05),IRE1 mRNA以及Caspase-12、CHOP蛋白表达水平明显下降(P <0.05,P <0.01),但ATF6mRNA表达水平差异无统计学意义(P>0.05)。结论高浓度葡萄糖和CORT能降低应激损伤状态糖尿病RINm5f细胞的活性,升高INS水平,加重胰岛素抵抗(IR)程度。逍遥散含药血清能缓解细胞IR水平,其作用机制可能与调控内质网应激(ERS)相关。
Objective To study the effect of Xiaoyaosan drug-containing serum on the activity and insulin level of RIN-m5 f cells and its intervention mechanism. Methods RIN-m5 f cells were cultured for 12 h in high concentrations of glucose and corticosterone(CORT)to induce diabetic model cells in a stress-damaged state,and then were intervened using 30% Xiaoyaosan-containing serum. RIN-m5 f cells were divided into 7 groups,included normal group,G group,GCo group,GC group,GX group,GCoC group,and GCoX group. After 12 hours of intervention,cell viability and INS secretion were measured and compared. QPCR was used to detect the expression of ATF6 and IRE1 mRNAs in each group. Western Blot was used to detect the expression of Caspase-12 and CHOP proteins. Results Compared with normal group,cell viability,mRNA levels of ATF6 and IRE1,and protein levels of Caspase-12 and CHOP of G group cells were significantly decreased(P < 0.05,P < 0.01);of which the INS content was increased(P < 0.01). Compared with G group,cell viability of GCo group was significantly lower(P <0.01);the INS content,mRNA levels of ATF6 and IRE1,and protein levels of Caspase-12 and CHOP of G group cells were significantly upregulated(P < 0.05,P < 0.01). Compared with GC group,cell viability of the GX group was higher(P < 0.05);the mRNA levels of ATF6 and IRE1,and protein levels of Caspase-12 were significantly upregulated(P < 0.05,P < 0.01),with no obvious changes in INS and CHOP levels(P>0.05). Compared with GCoC group,cell viability and INS level of the GCoX group were higher(P < 0.05),the mRNA level of IRE1,and protein levels of Caspase-12 and CHOP were significantly downregulated(P < 0.05,P < 0.01),with no obvious changes in ATF6 mRNA level(P>0.05). Conclusion The high concentration of glucose and CORT can decrease the viability of RIN-m5 f cells, increase the INS level, and increase the degree of insulin resistance(IR).Xiaoyaosan drug-containing serum can relieve the level of cellular IR,and its mechanism may be related to the regulation of islet β-cell endoplasmic reticulum stress(ERS).
Objective To study the effect of Xiaoyaosan drug-containing serum on the activity and insulin level of RIN-m5 f cells and its intervention mechanism. Methods RIN-m5 f cells were cultured for 12 h in high concentrations of glucose and corticosterone(CORT)to induce diabetic model cells in a stress-damaged state,and then were intervened using 30% Xiaoyaosan-containing serum. RIN-m5 f cells were divided into 7 groups,included normal group,G group,GCo group,GC group,GX group,GCoC group,and GCoX group. After 12 hours of intervention,cell viability and INS secretion were measured and compared. QPCR was used to detect the expression of ATF6 and IRE1 mRNAs in each group. Western Blot was used to detect the expression of Caspase-12 and CHOP proteins. Results Compared with normal group,cell viability,mRNA levels of ATF6 and IRE1,and protein levels of Caspase-12 and CHOP of G group cells were significantly decreased(P < 0.05,P < 0.01);of which the INS content was increased(P < 0.01). Compared with G group,cell viability of GCo group was significantly lower(P <0.01);the INS content,mRNA levels of ATF6 and IRE1,and protein levels of Caspase-12 and CHOP of G group cells were significantly upregulated(P < 0.05,P < 0.01). Compared with GC group,cell viability of the GX group was higher(P < 0.05);the mRNA levels of ATF6 and IRE1,and protein levels of Caspase-12 were significantly upregulated(P < 0.05,P < 0.01),with no obvious changes in INS and CHOP levels(P>0.05). Compared with GCoC group,cell viability and INS level of the GCoX group were higher(P < 0.05),the mRNA level of IRE1,and protein levels of Caspase-12 and CHOP were significantly downregulated(P < 0.05,P < 0.01),with no obvious changes in ATF6 mRNA level(P>0.05). Conclusion The high concentration of glucose and CORT can decrease the viability of RIN-m5 f cells, increase the INS level, and increase the degree of insulin resistance(IR).Xiaoyaosan drug-containing serum can relieve the level of cellular IR,and its mechanism may be related to the regulation of islet β-cell endoplasmic reticulum stress(ERS).
引文
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[2]陈晶,李璟.糖尿病细胞模型及研究进展[J].临床与病理杂志,2016,36(4):507-514.
[3]MARSHAK S,LEIBOWITZ G,BERTUZZI F,et al.Impairedβ-cell functions induced by chronic exposure of cultured human pancreatic islets to high glucose[J].Diabetes,1999,48(6):1230-1236.
[4]丁静云,应颖,邬磊,等.INS-1E细胞葡萄糖毒性模型的建立[J].现代生物医学进展,2015,15(22):4244-4247.
[5]RAúL B G,ANTONIO F L,ARTURO B G,et al.Hyperglycemia promotes p53-Mdm2 interaction but reduces p53 ubiquitination in RINm5F cells[J].Mol Cell Biochem,2015,405(1-2):257-264.
[6]GRAF E N,WHEELER R A,BAKER D A,et al.Corticosterone acts in the nucleus accumbens to enhance dopamine signaling and potentiate reinstatement of cocaine seeking[J].Journal of Neuroscience,2013,33(29):11800-11810.
[7]黄巧玲,吴华丽,蔡旻煊,等.皮质酮与慢性不可预见性应激诱导的两种抑郁症模型比较[J].解剖学报,2017,48(3):273-281.
[8]周珺,张黎,席红领,等.逍遥散加减对糖尿病合并抑郁症治疗作用的Mate分析[J].中医学报,2017,32(233):1878-1882.
[9]李娜,刘群,李晓娟,等.逍遥散对2型糖尿病兼抑郁症模型大鼠的作用效果[J].中华中医药杂志,2015,30(6):1948-1952.
[10]HONG J,KIM K,KIM J H,et al.The role of endoplasmic reticulum stress in cardiovascular disease and exercise[J].Int J Vasc Med,2017,2017:1-9.
[11]CHROUSOS G P,GOLD P W.The concepts of stress and stress system disorders.Overview of Physical and Behavioral Homeostasis[J].JAMA,1992,267(9):1244-1452.
[12]CHAUDHARI N,TALWAR P,PARIMISETTY A,et al.Amolecular web:endoplasmic reticulum stress,inflammation,and oxidative stress[J].Front Cell Neurosci,2014,8:213.
[13]YANG L,ZHAO D,REN J,et al.Endoplasmic reticulum stress and protein quality control in diabetic cardiomyopathy[J].Biochim Biophys Acta,2015,1852(2):209-218.
[14]RON D,WALTER P.Signal integration in the endoplasmic reticulum unfolded protein response[J].Nat Rev Mol Cell Biol,2007,8(7):519-529.
[15]涂浩,周琴,张迅,等.竹节参总皂苷对棕榈酸诱导HepG2细胞凋亡的保护作用研究[J].中国中药杂志,2018,43(2):390-395.
[16]杨方万,穆茂瑗,肖娟娟,等.内质网应激诱导细胞凋亡机制的研究进展[J].综述与进展,2014,43(10):176-180.
[17]MCCULLOUGH K D,MARTINDALE J L,KLOTZ L O,et al.Cadd153 sensitizes cells to endoplasmic reticulum stress by downregulating Bcl2 and perturbing the cellular redox state[J].Mol Cell Biol,2001,21(4):1249-1259.
[18]NAKAGAWA T,ZHU H,MORISHIMA N,et al.Caspase-12mediates ER-specific apoptosis and cytotoxicity by amyloid beta[J].Nature,2000,403(6765):98-103.