1,25-二羟基维生素D_(3)对急性重症胰腺炎模型大鼠淋巴细胞凋亡及Toll样受体4和9表达的影响  

Effects of 1,25-dihydroxyvitamin D_(3) on lymphocytes apoptosis and the expression of Toll-like receptors 4 and 9 in rats with severe acute pancreatitis

作　　者：吴海涛[1,2] 石海霞 宋玉[1] 周洪礼[1] WU Hai-tao;SHI Hai-xia;SONG Yu;ZHOU Hong-li(Department of Emergency Medicine,Affiliated Hospital of Qinghai University,Xining 810001,China;Graduate School of Qinghai University,Xining 810000,China)

机构地区：[1]青海大学附属医院急诊内科,西宁810001 [2]青海大学研究生院,西宁810000

出　　处：《药物分析杂志》2021年第12期2063-2069,共7页Chinese Journal of Pharmaceutical Analysis

基　　金：2019年青海省卫生健康系统指导性计划课题(2019-wjzdx-63)。

摘　　要：目的:探究1,25-二羟基维生素D_(3)对急性重症胰腺炎模型大鼠淋巴细胞凋亡及Toll样受体4和9表达的影响。方法:选取90只大鼠,随机分为:正常对照组(A)、模型对照组(B)、低剂量组(C)、中剂量组(D)、高低剂量组(E)和阳性药对照组(F),每组15只。除A组外,其余各组使用牛磺胆酸钠制成急性重症胰腺炎大鼠模型;C、D、E组分别腹腔注射2.5、5、10μg·kg^(-1)的1,25-二羟基维生素D_(3),F组皮下注射40 mg·kg^(-1)的奥曲肽;采用HE染色观察胰腺组织病理损伤,免疫组化检测胰腺组织的Caspase3含量,实时荧光定量PCR和蛋白质印迹检测淋巴细胞Caspase 3和Caspase 9 mRNA和蛋白的表达,酶联免疫吸附法结合RT-PCR检测一氧化氮合成酶(iNOS)、白介素(IL-6、IL-4及IL-10)、肿瘤坏死因子α(TNF-α)水平,蛋白质印迹法检测大鼠胰腺组织中Toll样受体4(TLR4)、Toll样受体9(TLR9)及核因子κB(NF-κB)的磷酸化情况。结果:相比A组,B组大鼠Caspase3阳性细胞数、Caspase 9、Caspase 3 mRNA的相对水平、cleaved Cas9/Cas9和cleaved Cas3/Cas3值、iNOS、IL-6、IL-4、IL-10及TNF-α表达、TLR4蛋白相对表达、TLR9蛋白相对表达及p65磷酸化水平均显著升高(P<0.05);相比B、D、E和F组Caspase3阳性细胞数、Caspase 9、Caspase 3 mRNA的相对水平、cleaved Cas9/Cas9、cleaved Cas3/Cas3值、iNOS、IL-6及TNF-α表达、TLR4蛋白相对表达、TLR9蛋白相对表达及p65磷酸化水平均显著降低,IL-4、IL-10表达显著升高(P<0.05)。结论:1,25-二羟基维生素D_(3)可抑制重症胰腺炎大鼠淋巴细胞凋亡、缓解炎症反应,可能与抑制TLR4、9表达有关。Objective:To explore the effects of 1,25-dihydroxyvitamin D_(3)[1,25(OH)_(2)D_(3)]on lymphocytesapoptosis and the expression of Toll-like receptors(TLR)4 and 9 in rats with severe acute pancreatitis(SAP).Methods:90 rats were randomly divided into 5 groups with 15 rats in each:normal control group(A),model control group(B),low-dose group(C),medium-dose group(D),high-dose group(E)and positive drug control group(F).Except for group A,the rats were made into SAP models with sodium taurocholate in the other groups.Groups C,D and E were intraperitoneally injected with2.5,5 and 10μg·kg^(-1)1,25(OH)_(2)D_(3),respectively.The group F was subcutaneously injected with 40 mg·kg^(-1)octreotide.The pathological damage in pancreatic tissues was observed by HE staining.The content of Caspase 3 in pancreatic tissues was detected by immunohistochemistry.The mRNA and proteins expression of Caspase 3 and Caspase 9 were detected by real-time fluorescent quantitative PCR and Western blot.The levels of inducible nitric oxide synthase(iNOS),interleukins(IL-6,IL-4,IL-10)and tumor necrosis factor-α(TNF-α)were detected by enzyme-linked immunosorbent assay and RT-PCR.The expression of TLR4 and TLR9,and phosphorylation of nuclear factorκB(NF-κB)in pancreatic tissues were detected by Western blot.Results:Compared with group A,the number of Caspase 3 positive cells,the relative levels of Caspase 9 and Caspase 3 mRNA,the values of cleaved Cas9/Cas9 and cleaved Cas3/Cas3,the expression of iNOS,IL-6,IL-4,IL-10 and TNF-α,the relative expression of TLR4 and TLR9 proteins,and phosphorylation level of p65 were significantly increased in group B(P<0.05).Compared with group B,the number of caspase-3 positive cells,the relative levels of Caspase 9 and Caspase 3 mRNA,the values of cleaved Cas9/Cas9 and cleaved Cas3/Cas3,the expression of iNOS,IL-6 and TNF-α,the relative expression of TLR4 and TLR9 proteins,and the phosphorylation level of p65 were significantly decreased in groups D,E and F,while the expressions of IL-4 and IL-10 were significan

关 键 词：重症胰腺炎 维生素D_(3) 淋巴细胞 细胞凋亡 炎症反应 荧光定量 酶联免疫吸附法 蛋白质印迹法 

分 类 号：R917[医药卫生—药物分析学]