8—O—乙酰山栀子苷甲酸对慢性炎性痛模型大鼠脊髓背角内HDAC1～5表达的影响及与JAK2—STAT3信号通路的关系研究(1)
中圖分类号 R285；R441.1 文献标志码 A 文章编号 1001-0408(2019)05-0608-06
DOI 10.6039/j.issn.1001-0408.2019.05.07
摘 要 目的：研究8-O-乙酰山栀子苷甲酸(8-OaS)对慢性炎性痛模型大鼠脊髓背角内组蛋白去乙酰化酶1～5(HDAC1～5)表达的影响及与Janus激酶2-信号转导与转录活化因子3(JAK2-STAT3)信号通路的关系。方法：取SD大鼠随机分为正常对照组、假手术组(生理盐水)、完全弗氏佐剂(CFA)组(生理盐水)和8-OaS低、中、高剂量组(2、20、200 μg/kg)，每组6只，除正常对照组和假手术组外，其余各组大鼠左侧后足趾侧皮下注射CFA复制慢性炎性痛模型，建模后腹腔注射相应药物，每日1次，连续7 d。采用热辐射法检测首次给药第1、2、3、4、5、6、7、8、11、15天大鼠的缩足潜伏期。另取大鼠按上述后5组方法进行分组给药，采用Western blot法检测大鼠末次给药后腰膨大节段脊髓背角中HDAC1～5、磷酸化JAK2(pJAK2)、磷酸化STAT3(pSTAT3)蛋白表达情况。再另取大鼠随机分为假手术组(生理盐水)、CFA组(生理盐水)、8-OaS组(20 μg/kg)和JAK2-STAT3的拮抗药α-氰基-(3，4-羟基)-N-苄苯乙烯胺(AG490)组(8 mg/kg)，每组6只，同上法复制IP模型后腹腔注射相应药物，每日1次，连续7 d，采用免疫荧光组织化学染色观察各组大鼠HDAC5和胶质纤维酸性蛋白(GFAP)在脊髓背角的表达情况。结果：与正常对照组和假手术组比较，其余各组大鼠的缩足潜伏期均明显缩短(P<0.05)；与CFA组比较，8-OaS低、中、高剂量组大鼠的缩足潜伏期均明显延长(P<0.05)，且呈剂量依赖性。与假手术组比较，CFA组大鼠脊髓背角中HDAC1～5、pJAK2、pSTAT3蛋白表达均明显增强(P<0.05)；与CFA组比较，8-OaS低、中、高剂量组大鼠脊髓背角中HDAC5、pJAK2、pSTAT3蛋白表达均明显降低(P<0.05)，但HDAC1～4蛋白表达差异均无统计学意义(P>0.05)。HDAC5大量表达于星形胶质细胞上，与假手术组比较，CFA组大鼠脊髓背角中GFAP和HDAC5表达均明显增强(P<0.05)；与CFA组比较，8-OaS组和AG490组大鼠脊髓背角中GFAP和HDAC5表达均明显降低(P<0.05)。结论：8-OaS可有效缓解由CFA诱导的慢性炎性痛，其机制可能与下调脊髓背角中HDAC5表达和JAK2、STAT3的磷酸化水平有关。
关键词 8-O-乙酰山栀子苷甲酸；慢性炎性痛；组蛋白去乙酰化酶；星形胶质细胞；Janus激酶2-信号转导与转录活化因子3；大鼠
Study on the Effects of 8-O-acetyl-shanzhiside Methylester on the Expression of HDAC 1-5 in the Spinal Dorsal Horn of Rats with Chronic Inflammatory Pain and Its Relationship with JAK2-STAT3 Signaling Pathway
WANG Jian1，2，XIAO Xiaoli3，CUI Jia1，WANG Lei1，FAN Tingting1，ZHANG Wei1(1.Dept. of Pharmacy， the First Affiliated Hospital of Air Force Military Medical University， Xi’an 710032， China；2.Dept. of Ambulatorium， No. 94750 Military Hospital of PLA， Fujian Liancheng 366200， China；3.Dept. of Rheumatology， the Affiliated TCM Hospital of Shanghai University of TCM， Shanghai 200071， China)
ABSTRACT OBJECTIVE： To study the effects of 8-O-acetyl-shanzhiside methylester (8-OaS) on the expression of histone deacetylase 1-5 (HDAC1-5) in the spinal dorsal horn of chronic inflammatory pain model rats， and its relationship with Janus-activated kinase 2-signal transductions and activators of transcription 3 (JAK2-STAT3) signaling pathway. METHODS： SD rats were randomly divided into normal control group， sham operation group (normal saline)， complete Freund’s adjuvant (CFA) group (normal saline)， 8-OaS low-dose， medium-dose and high-dose groups (2， 20， 200 μg/kg)， with 6 rats in each group. Except for normal control group and sham operation group， chronic inflammatory pain model was induced by subcutaneous injection of CFA into the left hind toe of rats in other groups； after modeling， those groups were given relevant medicine intraperitoneally， once a day， for consecutive 7 d. Thermal radiation method was used to detect the latency of paw withdraw in rats on the 1st， 2nd， 3rd， 4th， 5th， 6th， 7th， 8th， 11th and 15th day of administration. Rats were grouped and given medicine according to the above method of the latter 5 groups. The protein expression of HDAC 1-5， phosphorylated JAK2 (pJAK2) and phosphorylated STAT3 (pSTAT3) in the spinal dorsal horn of lumbar enlargement segment in rats were detected by Western blot method after last medication. Rats were randomly divided into sham operation group (normal saline)， CFA group (normal saline)， 8-OaS group (20 μg/kg) and JAK2-STAT3 inhibtor AG490 group (8 mg/kg)， with 6 rats in each group； IP model was established by same method as above and then were given relevant medicine intraperitoneally， once a day， for consecutive 7 d. The expression of HDAC5 and glial fibrillary acidic protein (GFAP) in spinal dorsal horn of rats were detected by immunofluorescence histochemical staining. RESULTS： Compared with normal control group and sham operation group， the latency of paw withdraw was shortened significantly in other groups (P<0.05). Compared with CFA group， the latency of paw withdraw was prolonged significantly in 8-OaS low-dose， medium-dose and high-dose groups (P<0.05)， in dose-dependent manner. Compared with sham operation group， the protein expression of HDAC 1-5， pJAK2 and pSTAT3 in spinal dorsal horn of rats were increased significantly in CFA group (P<0.05). Compared with CFA group， the protein expression of HDAC5， pJAK2 and pSTAT3 in spinal dorsal horn of rats were decreased significantly in 8-OaS low-dose， medium-dose and high-dose groups (P<0.05)， but there was no statistical significance in the protein expression of HDAC 1-4 (P>0.05). HDAC5 was expressed on astrocytes in the spinal dorsal horn； compared with sham operation group， the expression of GFAP and HDAC 5 were increased significantly in spinal dorsal horn of rats in CFA group (P<0.05). Compared with CFA group， the expression of GFAP and HDAC5 in spinal dorsal horn of rats were decreased significantly in 8-OaS group and AG490 group (P<0.05). CONCLUSIONS： 8-OaS can effectively relieve CFA-induced chronic inflammatory pain， the mechanism of which may be associated with the down-regulation of HDAC5 expression and the phosphorylation levels of JAK2 and STAT3., 百拇医药(王健 肖小莉 崔佳 王磊 樊婷婷 张维)
DOI 10.6039/j.issn.1001-0408.2019.05.07
摘 要 目的：研究8-O-乙酰山栀子苷甲酸(8-OaS)对慢性炎性痛模型大鼠脊髓背角内组蛋白去乙酰化酶1～5(HDAC1～5)表达的影响及与Janus激酶2-信号转导与转录活化因子3(JAK2-STAT3)信号通路的关系。方法：取SD大鼠随机分为正常对照组、假手术组(生理盐水)、完全弗氏佐剂(CFA)组(生理盐水)和8-OaS低、中、高剂量组(2、20、200 μg/kg)，每组6只，除正常对照组和假手术组外，其余各组大鼠左侧后足趾侧皮下注射CFA复制慢性炎性痛模型，建模后腹腔注射相应药物，每日1次，连续7 d。采用热辐射法检测首次给药第1、2、3、4、5、6、7、8、11、15天大鼠的缩足潜伏期。另取大鼠按上述后5组方法进行分组给药，采用Western blot法检测大鼠末次给药后腰膨大节段脊髓背角中HDAC1～5、磷酸化JAK2(pJAK2)、磷酸化STAT3(pSTAT3)蛋白表达情况。再另取大鼠随机分为假手术组(生理盐水)、CFA组(生理盐水)、8-OaS组(20 μg/kg)和JAK2-STAT3的拮抗药α-氰基-(3，4-羟基)-N-苄苯乙烯胺(AG490)组(8 mg/kg)，每组6只，同上法复制IP模型后腹腔注射相应药物，每日1次，连续7 d，采用免疫荧光组织化学染色观察各组大鼠HDAC5和胶质纤维酸性蛋白(GFAP)在脊髓背角的表达情况。结果：与正常对照组和假手术组比较，其余各组大鼠的缩足潜伏期均明显缩短(P<0.05)；与CFA组比较，8-OaS低、中、高剂量组大鼠的缩足潜伏期均明显延长(P<0.05)，且呈剂量依赖性。与假手术组比较，CFA组大鼠脊髓背角中HDAC1～5、pJAK2、pSTAT3蛋白表达均明显增强(P<0.05)；与CFA组比较，8-OaS低、中、高剂量组大鼠脊髓背角中HDAC5、pJAK2、pSTAT3蛋白表达均明显降低(P<0.05)，但HDAC1～4蛋白表达差异均无统计学意义(P>0.05)。HDAC5大量表达于星形胶质细胞上，与假手术组比较，CFA组大鼠脊髓背角中GFAP和HDAC5表达均明显增强(P<0.05)；与CFA组比较，8-OaS组和AG490组大鼠脊髓背角中GFAP和HDAC5表达均明显降低(P<0.05)。结论：8-OaS可有效缓解由CFA诱导的慢性炎性痛，其机制可能与下调脊髓背角中HDAC5表达和JAK2、STAT3的磷酸化水平有关。
关键词 8-O-乙酰山栀子苷甲酸；慢性炎性痛；组蛋白去乙酰化酶；星形胶质细胞；Janus激酶2-信号转导与转录活化因子3；大鼠
Study on the Effects of 8-O-acetyl-shanzhiside Methylester on the Expression of HDAC 1-5 in the Spinal Dorsal Horn of Rats with Chronic Inflammatory Pain and Its Relationship with JAK2-STAT3 Signaling Pathway
WANG Jian1，2，XIAO Xiaoli3，CUI Jia1，WANG Lei1，FAN Tingting1，ZHANG Wei1(1.Dept. of Pharmacy， the First Affiliated Hospital of Air Force Military Medical University， Xi’an 710032， China；2.Dept. of Ambulatorium， No. 94750 Military Hospital of PLA， Fujian Liancheng 366200， China；3.Dept. of Rheumatology， the Affiliated TCM Hospital of Shanghai University of TCM， Shanghai 200071， China)
ABSTRACT OBJECTIVE： To study the effects of 8-O-acetyl-shanzhiside methylester (8-OaS) on the expression of histone deacetylase 1-5 (HDAC1-5) in the spinal dorsal horn of chronic inflammatory pain model rats， and its relationship with Janus-activated kinase 2-signal transductions and activators of transcription 3 (JAK2-STAT3) signaling pathway. METHODS： SD rats were randomly divided into normal control group， sham operation group (normal saline)， complete Freund’s adjuvant (CFA) group (normal saline)， 8-OaS low-dose， medium-dose and high-dose groups (2， 20， 200 μg/kg)， with 6 rats in each group. Except for normal control group and sham operation group， chronic inflammatory pain model was induced by subcutaneous injection of CFA into the left hind toe of rats in other groups； after modeling， those groups were given relevant medicine intraperitoneally， once a day， for consecutive 7 d. Thermal radiation method was used to detect the latency of paw withdraw in rats on the 1st， 2nd， 3rd， 4th， 5th， 6th， 7th， 8th， 11th and 15th day of administration. Rats were grouped and given medicine according to the above method of the latter 5 groups. The protein expression of HDAC 1-5， phosphorylated JAK2 (pJAK2) and phosphorylated STAT3 (pSTAT3) in the spinal dorsal horn of lumbar enlargement segment in rats were detected by Western blot method after last medication. Rats were randomly divided into sham operation group (normal saline)， CFA group (normal saline)， 8-OaS group (20 μg/kg) and JAK2-STAT3 inhibtor AG490 group (8 mg/kg)， with 6 rats in each group； IP model was established by same method as above and then were given relevant medicine intraperitoneally， once a day， for consecutive 7 d. The expression of HDAC5 and glial fibrillary acidic protein (GFAP) in spinal dorsal horn of rats were detected by immunofluorescence histochemical staining. RESULTS： Compared with normal control group and sham operation group， the latency of paw withdraw was shortened significantly in other groups (P<0.05). Compared with CFA group， the latency of paw withdraw was prolonged significantly in 8-OaS low-dose， medium-dose and high-dose groups (P<0.05)， in dose-dependent manner. Compared with sham operation group， the protein expression of HDAC 1-5， pJAK2 and pSTAT3 in spinal dorsal horn of rats were increased significantly in CFA group (P<0.05). Compared with CFA group， the protein expression of HDAC5， pJAK2 and pSTAT3 in spinal dorsal horn of rats were decreased significantly in 8-OaS low-dose， medium-dose and high-dose groups (P<0.05)， but there was no statistical significance in the protein expression of HDAC 1-4 (P>0.05). HDAC5 was expressed on astrocytes in the spinal dorsal horn； compared with sham operation group， the expression of GFAP and HDAC 5 were increased significantly in spinal dorsal horn of rats in CFA group (P<0.05). Compared with CFA group， the expression of GFAP and HDAC5 in spinal dorsal horn of rats were decreased significantly in 8-OaS group and AG490 group (P<0.05). CONCLUSIONS： 8-OaS can effectively relieve CFA-induced chronic inflammatory pain， the mechanism of which may be associated with the down-regulation of HDAC5 expression and the phosphorylation levels of JAK2 and STAT3., 百拇医药(王健 肖小莉 崔佳 王磊 樊婷婷 张维)
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