慢性脑缺血大鼠rhEPO经鼻给药激活JAK2/STAT3通路及对Bcl-2和Bax表达的影响

Activating JAK2 / STAT3 pathway by rhEPO via intranasal administration and its impact on Bcl-2 and Bax in rats with chronic cerebral ischemia

目的探讨重组人促红细胞生成素(rh EPO)对大鼠慢性脑缺血后JAK2/STAT3信号传导通路的影响,以及对Bcl-2和Bax表达的影响,分析rh EPO在慢性脑缺血中可能的作用机制。方法将60只雄性成年SD大鼠,随机分为假手术组、2VO组(双侧颈总动脉结扎模型组)、2VO+rh EPO组、2VO+rh EPO+AG490(JAK2特异性拮抗剂)组、2VO+AG490组,每组12只。各组分别腹腔注射AG490(5 mg/kg)或等量的DMSO(AG490的溶媒),30 min后鼻腔缓慢注入rh EPO(150 U/125μl)或等量生理盐水。造模后3 d给药,每周1次,共8次。给药毕24 h后取脑组织标本,HE染色观察组织形态学变化。免疫组化法检测JAK2、P-JAK2(Tyr1007 1008)、STAT3、P-STAT3(Tyr705),以及Bcl-2、Bax蛋白的表达水平。原位末端标记法(TUNEL)检测凋亡细胞数。q RT-PCR法检测Bcl-2、Bax的m RNA表达水平。结果 (1)与假手术组比较,2VO组中P-JAK2和P-STAT3表达增强,Bcl-2、Bax的表达上调(P均<0.05)。(2)与2VO组比较,2VO+rh EPO组P-JAK2、P-STAT3蛋白表达增强,并上调Bcl-2的表达和下调Bax的表达(P均<0.05)。(3)与2VO+rh EPO组比较,2VO+rh EPO+AG490组可抑制rh EPO诱导的P-JAK2、P-STAT3表达,下调Bcl-2表达和上调Bax表达(P均<0.05)。(4)2VO+AG490组中P-JAK2、P-STAT3、Bcl-2、Bax的表达与2VO组和2VO+rh EPO+AG490组比较差异无统计学意义(P均>0.05)。结论 rh EPO可能通过激活JAK2/STAT3信号转导通路,促进Bcl-2上调和Bax下调,减缓慢性脑缺血大鼠神经细胞凋亡。

Objective To study the effects of recombinant human erythropoietin（ rh EPO） on JAK2 / STAT3 pathway,an anti-apoptosis signal pathway,and Bcl-2 and Bax expressions after chronic cerebral ischemia（ CCI） in rats and analyze the potential acting mechanism of rh EPO in CCI. Methods Sixty healthy male SD rats were randomly divided into five groups（n = 12 each）：sham-operated group,bilateral common carotid artery ligation model group（2VO group）,2VO ＋ rh EPO group,2VO ＋ rh EPO ＋ AG490（ specific antagonist of JAK2） group,2VO ＋ AG490 group. AG490（5 mg / kg） or an equal amount of DMSO（ solvent of AG490） was respectively given by intraperitoneal injection,then rh EPO（150 U /125 μl） or an equal amount of normal saline was respectively given via nasal cavity following 30 minutes in different groups. The drugs were weekly administered three days after molding for eight times. The brain tissue samples were taken 24 hours after the last medication. The morphological changes of brain tissues were observed by HE staining;JAK2,P-JAK2（ Tyr1007 1008）,STAT3,P-STAT3（ Tyr705） and the expression levels of Bcl-2 and Bax proteins were detected by immunnohistochemistry;the number of apoptotic cells was detected by terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling assay（ TUNEL）;the expression levels of Bcl-2 m RNA and Bax m RNA were measured by real time quantity RT-PCR（q RT-PCR）. Results（1） Compared with sham-operated group,the expressions of P-JAK2 and P-STAT3 increased and the expressions of Bcl-2 and Bax were up-regulated in 2VO group（ all P〈0. 05）.（2） Compared with 2VO group,the expressions of P-JAK2 and P-STAT3 increased;the expression of Bcl-2 was up-regulated;the expression of Bax was down-regulated in 2VO ＋ rh EPO group（ all P〈0. 05）.（3） Compared with 2VO ＋ rh EPO group,rh EPO-induced expressions of PJAK2 and P-STAT3 decreased;expression of Bcl-2 was down-regulated;expression of Bax was up-regulated in 2VO ＋ rh EPO ＋ AG490 group（ all P〈0. 05）.（4） The 2VO ＋ AG490 group was similar with 2VO group and 2VO ＋ rh EPO ＋ AG490 group in the expressions of P-JAK2,P-STAT3,Bcl-2 and Bax（ all P〈0. 05）. Conclusion The action of attenuating nerve cells apoptosis of rh EPO in CCI rats might be by activating the signal transduction of JAK2 / STAT3 and promoting up-regulation of Bcl-2 and down-regulation of Bax.