电针对脑缺血再灌注模型大鼠炎性反应相关信号的影响及机制研究
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摘要
研究背景：
中风具有高发病率、高致残率、高复发率,仅中国每年便新增150-200万病例,其中,缺血性脑中风为最常见中风类型,占全部中风的60-80%。越来越多研究表明,炎症过程参与中风后损伤级联反应,可能是造成脑缺血后再灌注损伤的主要原因。早期溶栓、抗凝治疗为循证医学确认的有效治疗方法,但存在严格的时间窗限制,在长期恢复过程中效果不显著。
大量临床资料和实验研究表明,针刺为治疗缺血性脑中风的有效手段,急性期、恢复期、后遗症及康复期均有显著效果。
研究目的：
本课题通过观察针刺干预脑缺血再灌注模型大鼠后,中枢损伤局部、对侧脑组织和外周血中应激-损伤-修复信号链中关键信号的动态变化,从信号传导链、病程发展过程、机体功能整体调控的综合角度,探讨针刺治疗脑缺血再灌注后炎症损伤的机制,用现代生物医学研究手段,阐明针灸对损伤-应激与损伤-修复相关疾病过程的作用机制,为临床治疗方案提供确切的实验室依据。
研究方法：
1、将大鼠按随机数字表分成空白对照组、假手术组和模型对照组,每组10只动物。对造模后动物进行神经功能评分,采用TTC染色观察大脑梗死情况,HE染色观察脑组织形态学变化。
2、将大鼠按随机数字表分成空白对照组(10只动物)、假手术组(10只动物)和造模组,造模成功(根据神经功能评分)的大鼠根据随机数字表又分为模型对照组、尼莫地平组、针刺治疗组(百会透刺印堂配伍人中穴),共4组,每组又分为12h、24h、48h.72h.96h.144h6个时程,每个时程10只动物。取外周血清,ELISA(?)去检测炎性反应相关应激信号-ACTH,内源性危险信号-Hsp70,启动和调节信号-IL-6,IL-1β,损伤效应信号-TNFα的含量；取脑组织,冰冻切片,免疫组化检测患侧与健侧脑组织中炎性反应相关信号：启动和调节信号-IL-6、IL-1β,损伤效应信号-TNFα,MMP-9,局部炎症损伤相关粘附、趋化因子-ICAM-1,MCP-1,以及修复信号-TGFβ的表达。
3、将大鼠按随机数字表分成空白对照组(10只动物)和造模组,造模成功(根据神经功能评分)的大鼠根据随机数字表又分为模型对照组、针刺治疗1组(百会透刺印堂配伍人中穴)、针刺治疗2组(百会配伍患侧足三里穴),共4组,每组又分为12h、24h、48h、72h、96h、144h6个时程,每个时程10只动物。取取脑组织,冰冻切片免疫组化检测患侧组织中IL-6,TNFα,MCP-1,TGFβ的表达。
研究结果：
1、模型评价：
TTC染色发现缺血局部脑组织出现典型染色缺失,呈现白色,与大脑中动脉支配区域相符,视交叉前后梗死灶比较稳定。
HE染色镜下观察发现,梗死中心区染色变浅,细胞数目减少,细胞核固缩,核仁变小,形态不规则；问质水肿；内皮细胞肿胀；可以见到大量网格状空泡。
2、针刺对血清中炎性反应相关信号的影响：
脑缺血再灌注模型大鼠血清中炎性反应相关信号ACTH,IL-1β,IL=6,TNF α,Hsp70,在损伤发展过程中呈现典型双峰曲线模式,且各信号变化趋势相似。ACTH在12h到达第一峰峰值,在48h达第二.峰峰值；IL-1β, IL-6, TNFα, Hsp70均在24h到达第一峰峰值,在72-96h达第二峰峰值。假手术组ACTH,IL-6,TNFα,Hsp70呈现相似双峰表达趋势,IL-1β仅72h高于生理水平。
给予百会透刺印堂配伍人中电针治疗后,可显著下调外周血请中炎性反应信号ACTH,IL-1β,IL-6,TNF α, Hsp70的第一峰表达水平；显著下调ACTH,IL-1β,TNFα的第二峰表达水平;上调1L-6的第二峰表达水平。
3、针刺对脑组织中炎性反应相关信号的影响：
脑缺血再灌注模型大鼠患侧脑组织中炎性反应相关信号IL-6,IL-1β,MMP-9, ICAM-1,MCP-1,TGFβ,在损伤发展过程中呈现典型双峰曲线模式；均在12-24h到达第一峰峰值;IL-6在96h时到达第二峰峰值；IL-1β,MMP-9在48h即到达第二峰峰值；ICAM-1,MCP-1,TGFβ在72h到达第二峰峰值；TNFα第一峰于第二峰融合,12h开始持续增高,24h到达峰值,保持较高水平至72h。假手术组呈现相似表达趋势。
脑缺血再灌注模型大鼠健侧脑组织中炎性反应相关信号均呈现典型双峰信号IL-6,IL-1β,TNFα,ICAM-1,MCP-1,TGFβ在12-24h到达第一峰峰值；IL-6在96h时到达第二峰峰值；IL-1β自48h持续增高,144h时到达最高值；TNFα在48h到达第一峰峰值；ICAM-1,MCP-1,TGFβ在72h到达第二峰峰值；MMP-9在48h达到第一峰峰值,自72h持续增高,144h到达最高值。假手术组呈现相似表达趋势。
模型大鼠给予百会透刺印堂配伍人中电针治疗后,对双侧脑组织中炎性反应相关信号的影响是：对患侧脑组织中IL-6,IL-1β,TNFα,MMP-9,MCP-1,TGFβ的表达水平均有全程下调作用；对健侧脑组织中IL-6,MMP-9,MCP-1,TGFβ的表达水平均有全程下调作用；对健侧脑组织中IL-6,MMP-9损伤急性期(48h)的表达水平有上调作用；而针刺治疗组ICAM-1的表达在患侧脑组织中72h前呈增高趋势,在健侧脑组织中48h前呈增高趋势。
针刺治疗后,患侧脑组织中IL-6,ICAM-1第二峰的表达提前出现；健侧脑组织中,IL-6,IL-1β,MMP-9,ICAM-1,MCP-1,TGFβ第二峰的表达提前出现。
4、针刺不同穴组对患侧脑组织中炎性反应相关信号的影响：
百会穴配伍足三里穴电针治疗,对比选取百会透刺印堂配伍人中穴,能够显著下调模型大鼠患侧脑组织中炎性反应相关信号：IL-6,TNFα,MCP-1,TGFβ的全程表达水平和第一峰、第二峰的峰值；IL-6,TGFβ第二峰的表达提前。
研究结论：
1、血管内栓阻断线法脑缺血再灌注大鼠模型,造成大脑中动脉供血区域的梗塞并产生功能障碍,且具有一定的稳定性；脑缺血再灌注损伤启动了机体局部与整体,包括外周血清、双侧脑组织中炎性／免疫相关的应激-损伤-修复信号链；模型复制成功。
2、电针百会透刺印堂配伍人中穴对机体应激-损伤-修复信号链发挥网络调节作用,整体影响了外周血清、双侧脑组织中炎性反应相关应激信号、内源性危险信号、启动和调节信号、损伤效应信号、联络信号、修复信号的表达；电针可能通过抑制机体过度应激、减轻炎性损伤过程、促进细胞间联系、提前启动修复功能、调动健侧脑组织进行调节代偿减轻缺血再灌注损伤。
3、百会配伍足三里穴对比百会透刺印堂配伍人中穴,能够更好调节机体对炎症应激、损伤的反应,减轻炎性反应及炎症损伤,更早启动修复功能；提示头部穴位、肢体穴位联合运用干预炎症损伤的效应优于单纯头部取穴。
Background:
Stroke is the common and frequently-occurring disease overworld which with high incidence, high morbidity and high recurrence rate. There are150-200million new cases each year in China. Ischemic stroke is the most common type of stroke, accounting for60-80%of all stroke. More and more studies show that the inflammatory process involved in stroke damage cascade, the main reason may be caused by cerebral ischemia and reperfusion injury. Early thrombolysis, anticoagulation therapy are the effective treatment recognized by evidence-based medicine, but there is strict time window, the effect was not significant in the long-term recovery process.
A large number of clinical and experimental studies have shown that acupuncture is an effective treatment to ischemic stroke, and have shown significant effect in acute, recovery, sequelae and rehabilitation phase.
Objectives:
The subject observed the the dynamic change of stress-damage-repair signal chain in the peripheral blood seram and ipsilateral/peripheral brain tissue after acupuncture intervention on cerebral ischemia and reperfusion model rats. To explore acupuncture treatment's precise mechanisms in cerebral ischemia and reperfusion inflammatory injury. To provide the esact laboratory evidence for clinical treatment programs.
Methods:
1, The rats were randomly divided into the control group, sham group and model control group,,10animals of each group. The model animal were evaluated by Neurological score, TTC staining(to observe cerebral infarction), and HE staining(to observe the morphological changes of the brain tissue).
2, The rats were randomly divided into the control group (10animals), sham group (10animals) and model group, model animals were divided into model control group, nimodipine group, acupuncture group(Baihui, Yintang and Renzhong) according to the random number table, each group was divided into6schedule groups-12h,24h,48h,72h,96h,144h,10animals of each schedule. Chose ELISA method to detect the inflammatory response related signals in serum, including stress signal-ACTH, endogenous danger signal-HSP70, start-up and adjustment signals-IL-6, IL-1β, signal of damage effect-TNFα; chose the immunohistochemical technology to detect the expression of inflammatory response signals in ipsilateral and contralateral brain tissue, including activate and adjust signals-IL-6, IL-1c, signal of damage effect-TNFα, MMP-9, contact signal-ICAM-1, MCP-1, and the repair signal-TGFp.
3, The rats were randomly divided into the control group (10animals) and model group, model rats were divided into model control group, acupuncture treatment groupl (Baihui, Yintang and Renzhong), and acupuncture treatment group2(Baihui, ipsilateral Zusanli a), according to the random number table, then divided into12h,24h,48h,72h,96h,144h,6schedule in each group,10animals of each schedule. Take brain tissue, frozen sections, used the immunohistochemical technology to detect the expression of IL-6, TNF-α, MCP-1, TGFβ in contralateral brain tissue.
Results:
1, Model Evaluation
The TTC staining found that there was typical dyeing missing in local ischemic brain tissue, appear white, in line with the disposable area of the middle cerebral artery,and the infarction was relatively stable before and after the optic chiasm. HE staining were observed that, staining shallow in the central farction area, decrease of the cells number, nuclear condensation, smaller and irregular of the nucleolus; interstitial edema; endothelial cell swelling; a large number of grid-like vacuole.
2, The influence of acupuncture treatment to inflammatory response signals in serum: The inflammatory response signals ACTH, IL-1β, IL-6, TNF-α, Hsp70, in cerebral ischemia and reperfusion model rats' serum, showed typical bimodal curve mode in the process of damage development, and the trend were similar. ACTH reached the first peak at12h, and reached the second peak at48h; IL-1β, IL-6, TNF-α, Hsp70reached the first peak at24h, an second peak-to-peak at72-96h. ACTH, IL-6, TNF-α, Hsp70of sham-operated group showed similar bimodal expression trend, IL-1βof sham-operated group was higher than physiological levels only at72h.
Electro-acupuncture treatment on Baihui, Yintang and Renzhong could significantly lowered the the first peak'expression levels of inflammatory response signals ACTH, IL-1β, IL-6, TNF-α, and Hsp70in peripheral serum; could significantly lowered the second peak' expression levels of ACTH, IL-1β, TNF-α; could increase the second peak' expression level of IL-6.
3, The influence of acupuncture treatment to inflammatory response signals in the brain tissue:
The inflammatory response signals in cerebral ischemia and reperfusion model rats' local brain tissue, IL-6, IL-1β, MMP-9, ICAM-1, MCP-1, TGFβshowed typical bimodal curve mode in the process of damage development, and the trend were similar, reached the first peak at12-24h; IL-6reached the second peak at96h; IL-1(3, MMP-9reached the second peak at48h; ICAM-1, MCP-1, TGF(3reached the second peak at72h; TNFαcontinued to increase at12h, reached rhe first peak at24h, maintain a high level to72h. Sham operation group showed similar expression trends.
The inflammatory response signals in cerebral ischemia and reperfusion model rats" contralateral brain tissue, IL-6, IL-1β, TNFα, ICAM-1, MCP-1, TGFp showed typical bimodal curve mode in the process of damage development, to reached the first peak at12-24h; IL-6reached the second peak at96h; IL-1βcontinued to increase from48h, reached the highest value atl44h; TNFareached the first peak at48h; ICAM-1, MCP-1, TGFβreached the second peak at72h; MMP-9reached the first peak at48h, continued to increase from72h, reached the highest value at144h. Sham operation group showed similar expression trends. Electro-acupuncture treatment on Baihui, Yintang and Renzhong had significantly influences on inflammatory response signals in bilateral brain tissue. For the ipsilatcral brain tissue, the expression levels of IL-6, IL-1β, TNFα, MMP-9, MCP-1, TGFβ were lowered; for the contralateral brain tissue, the expression levels of IL-6, MMP-9, MCP-1. TGFβwere lowered; in contralateral brain tissue, the expression levels of IL-6, MMP-9were raises in the acute damage phase at48h; the expression of ICAM-1showed a rising trend before72h in the ipsilateral brain tissue, and before48h in the contralateral brain tissue.
After acupuncture treatment, the second peak' expression of IL-6, ICAM-1in Ipsilateral brain tissue were in early appearance; the second peak' expression of IL-6, IL-1β, MMP-9, ICAM-1, MCP-1TGFβ in contralateral brain tissue were in early appearance.
4, The influence of different acupoints to inflammatory response signals in the local brain tissue: Electro-acupuncture treatment on Baihui, Zusanli, contrast the points Baihui, Yintang and Renzhong, could significantly lower the inflammatory response signals'expression levels in the ipsilateral brain tissue in the rat model, including IL-6, TNF-α, MCP-1, TGFβ, could show the second peak of IL-6, TGFβ in advance.
Conclusions:
1, The brain ischemia reperfusion rat model was stable; cerebral ischemia-reperfusion injury activated the inflammatory/immune-related stress-damage-repair signal chain in body part and the whole, including peripheral serum, bilateral brain tissue; this model was copied successfully.
2, Electro-acupuncture treatment on Baihui, Yintang and Renzhong played the network regulation on stress-damage-repair signal chain, had the overall impact to the inflammatory response signals in he peripheral serum and bilateral brain tissue, including stress signal, endogenous danger signal, start and regulate signals, damage effect signals contact signals, and repair signal; electro-acupuncture may reduce ischemia-reperfusion injury through inhibiting excessive stress, reducing the inflammatory injury, promoting inter-cell contact, starting the repair function in advance, adjusting the healthside brain tissue to compensatory.3, Electro-acupuncture treatment on Baihui, Zusanli, contrast the points Baihui, Yintang and Renzhong, had better influences in regulating the body's inflammatory stress response to injury, reducing the inflammatory response and inflammatory, activating repair function; combined use of acupoints on the head and body may be better in reducing inflammatory injury than using the head acupuncture points only.
中风具有高发病率、高致残率、高复发率,仅中国每年便新增150-200万病例,其中,缺血性脑中风为最常见中风类型,占全部中风的60-80%。越来越多研究表明,炎症过程参与中风后损伤级联反应,可能是造成脑缺血后再灌注损伤的主要原因。早期溶栓、抗凝治疗为循证医学确认的有效治疗方法,但存在严格的时间窗限制,在长期恢复过程中效果不显著。
大量临床资料和实验研究表明,针刺为治疗缺血性脑中风的有效手段,急性期、恢复期、后遗症及康复期均有显著效果。
研究目的：
本课题通过观察针刺干预脑缺血再灌注模型大鼠后,中枢损伤局部、对侧脑组织和外周血中应激-损伤-修复信号链中关键信号的动态变化,从信号传导链、病程发展过程、机体功能整体调控的综合角度,探讨针刺治疗脑缺血再灌注后炎症损伤的机制,用现代生物医学研究手段,阐明针灸对损伤-应激与损伤-修复相关疾病过程的作用机制,为临床治疗方案提供确切的实验室依据。
研究方法：
1、将大鼠按随机数字表分成空白对照组、假手术组和模型对照组,每组10只动物。对造模后动物进行神经功能评分,采用TTC染色观察大脑梗死情况,HE染色观察脑组织形态学变化。
2、将大鼠按随机数字表分成空白对照组(10只动物)、假手术组(10只动物)和造模组,造模成功(根据神经功能评分)的大鼠根据随机数字表又分为模型对照组、尼莫地平组、针刺治疗组(百会透刺印堂配伍人中穴),共4组,每组又分为12h、24h、48h.72h.96h.144h6个时程,每个时程10只动物。取外周血清,ELISA(?)去检测炎性反应相关应激信号-ACTH,内源性危险信号-Hsp70,启动和调节信号-IL-6,IL-1β,损伤效应信号-TNFα的含量；取脑组织,冰冻切片,免疫组化检测患侧与健侧脑组织中炎性反应相关信号：启动和调节信号-IL-6、IL-1β,损伤效应信号-TNFα,MMP-9,局部炎症损伤相关粘附、趋化因子-ICAM-1,MCP-1,以及修复信号-TGFβ的表达。
3、将大鼠按随机数字表分成空白对照组(10只动物)和造模组,造模成功(根据神经功能评分)的大鼠根据随机数字表又分为模型对照组、针刺治疗1组(百会透刺印堂配伍人中穴)、针刺治疗2组(百会配伍患侧足三里穴),共4组,每组又分为12h、24h、48h、72h、96h、144h6个时程,每个时程10只动物。取取脑组织,冰冻切片免疫组化检测患侧组织中IL-6,TNFα,MCP-1,TGFβ的表达。
研究结果：
1、模型评价：
TTC染色发现缺血局部脑组织出现典型染色缺失,呈现白色,与大脑中动脉支配区域相符,视交叉前后梗死灶比较稳定。
HE染色镜下观察发现,梗死中心区染色变浅,细胞数目减少,细胞核固缩,核仁变小,形态不规则；问质水肿；内皮细胞肿胀；可以见到大量网格状空泡。
2、针刺对血清中炎性反应相关信号的影响：
脑缺血再灌注模型大鼠血清中炎性反应相关信号ACTH,IL-1β,IL=6,TNF α,Hsp70,在损伤发展过程中呈现典型双峰曲线模式,且各信号变化趋势相似。ACTH在12h到达第一峰峰值,在48h达第二.峰峰值；IL-1β, IL-6, TNFα, Hsp70均在24h到达第一峰峰值,在72-96h达第二峰峰值。假手术组ACTH,IL-6,TNFα,Hsp70呈现相似双峰表达趋势,IL-1β仅72h高于生理水平。
给予百会透刺印堂配伍人中电针治疗后,可显著下调外周血请中炎性反应信号ACTH,IL-1β,IL-6,TNF α, Hsp70的第一峰表达水平；显著下调ACTH,IL-1β,TNFα的第二峰表达水平;上调1L-6的第二峰表达水平。
3、针刺对脑组织中炎性反应相关信号的影响：
脑缺血再灌注模型大鼠患侧脑组织中炎性反应相关信号IL-6,IL-1β,MMP-9, ICAM-1,MCP-1,TGFβ,在损伤发展过程中呈现典型双峰曲线模式；均在12-24h到达第一峰峰值;IL-6在96h时到达第二峰峰值；IL-1β,MMP-9在48h即到达第二峰峰值；ICAM-1,MCP-1,TGFβ在72h到达第二峰峰值；TNFα第一峰于第二峰融合,12h开始持续增高,24h到达峰值,保持较高水平至72h。假手术组呈现相似表达趋势。
脑缺血再灌注模型大鼠健侧脑组织中炎性反应相关信号均呈现典型双峰信号IL-6,IL-1β,TNFα,ICAM-1,MCP-1,TGFβ在12-24h到达第一峰峰值；IL-6在96h时到达第二峰峰值；IL-1β自48h持续增高,144h时到达最高值；TNFα在48h到达第一峰峰值；ICAM-1,MCP-1,TGFβ在72h到达第二峰峰值；MMP-9在48h达到第一峰峰值,自72h持续增高,144h到达最高值。假手术组呈现相似表达趋势。
模型大鼠给予百会透刺印堂配伍人中电针治疗后,对双侧脑组织中炎性反应相关信号的影响是：对患侧脑组织中IL-6,IL-1β,TNFα,MMP-9,MCP-1,TGFβ的表达水平均有全程下调作用；对健侧脑组织中IL-6,MMP-9,MCP-1,TGFβ的表达水平均有全程下调作用；对健侧脑组织中IL-6,MMP-9损伤急性期(48h)的表达水平有上调作用；而针刺治疗组ICAM-1的表达在患侧脑组织中72h前呈增高趋势,在健侧脑组织中48h前呈增高趋势。
针刺治疗后,患侧脑组织中IL-6,ICAM-1第二峰的表达提前出现；健侧脑组织中,IL-6,IL-1β,MMP-9,ICAM-1,MCP-1,TGFβ第二峰的表达提前出现。
4、针刺不同穴组对患侧脑组织中炎性反应相关信号的影响：
百会穴配伍足三里穴电针治疗,对比选取百会透刺印堂配伍人中穴,能够显著下调模型大鼠患侧脑组织中炎性反应相关信号：IL-6,TNFα,MCP-1,TGFβ的全程表达水平和第一峰、第二峰的峰值；IL-6,TGFβ第二峰的表达提前。
研究结论：
1、血管内栓阻断线法脑缺血再灌注大鼠模型,造成大脑中动脉供血区域的梗塞并产生功能障碍,且具有一定的稳定性；脑缺血再灌注损伤启动了机体局部与整体,包括外周血清、双侧脑组织中炎性／免疫相关的应激-损伤-修复信号链；模型复制成功。
2、电针百会透刺印堂配伍人中穴对机体应激-损伤-修复信号链发挥网络调节作用,整体影响了外周血清、双侧脑组织中炎性反应相关应激信号、内源性危险信号、启动和调节信号、损伤效应信号、联络信号、修复信号的表达；电针可能通过抑制机体过度应激、减轻炎性损伤过程、促进细胞间联系、提前启动修复功能、调动健侧脑组织进行调节代偿减轻缺血再灌注损伤。
3、百会配伍足三里穴对比百会透刺印堂配伍人中穴,能够更好调节机体对炎症应激、损伤的反应,减轻炎性反应及炎症损伤,更早启动修复功能；提示头部穴位、肢体穴位联合运用干预炎症损伤的效应优于单纯头部取穴。
Background:
Stroke is the common and frequently-occurring disease overworld which with high incidence, high morbidity and high recurrence rate. There are150-200million new cases each year in China. Ischemic stroke is the most common type of stroke, accounting for60-80%of all stroke. More and more studies show that the inflammatory process involved in stroke damage cascade, the main reason may be caused by cerebral ischemia and reperfusion injury. Early thrombolysis, anticoagulation therapy are the effective treatment recognized by evidence-based medicine, but there is strict time window, the effect was not significant in the long-term recovery process.
A large number of clinical and experimental studies have shown that acupuncture is an effective treatment to ischemic stroke, and have shown significant effect in acute, recovery, sequelae and rehabilitation phase.
Objectives:
The subject observed the the dynamic change of stress-damage-repair signal chain in the peripheral blood seram and ipsilateral/peripheral brain tissue after acupuncture intervention on cerebral ischemia and reperfusion model rats. To explore acupuncture treatment's precise mechanisms in cerebral ischemia and reperfusion inflammatory injury. To provide the esact laboratory evidence for clinical treatment programs.
Methods:
1, The rats were randomly divided into the control group, sham group and model control group,,10animals of each group. The model animal were evaluated by Neurological score, TTC staining(to observe cerebral infarction), and HE staining(to observe the morphological changes of the brain tissue).
2, The rats were randomly divided into the control group (10animals), sham group (10animals) and model group, model animals were divided into model control group, nimodipine group, acupuncture group(Baihui, Yintang and Renzhong) according to the random number table, each group was divided into6schedule groups-12h,24h,48h,72h,96h,144h,10animals of each schedule. Chose ELISA method to detect the inflammatory response related signals in serum, including stress signal-ACTH, endogenous danger signal-HSP70, start-up and adjustment signals-IL-6, IL-1β, signal of damage effect-TNFα; chose the immunohistochemical technology to detect the expression of inflammatory response signals in ipsilateral and contralateral brain tissue, including activate and adjust signals-IL-6, IL-1c, signal of damage effect-TNFα, MMP-9, contact signal-ICAM-1, MCP-1, and the repair signal-TGFp.
3, The rats were randomly divided into the control group (10animals) and model group, model rats were divided into model control group, acupuncture treatment groupl (Baihui, Yintang and Renzhong), and acupuncture treatment group2(Baihui, ipsilateral Zusanli a), according to the random number table, then divided into12h,24h,48h,72h,96h,144h,6schedule in each group,10animals of each schedule. Take brain tissue, frozen sections, used the immunohistochemical technology to detect the expression of IL-6, TNF-α, MCP-1, TGFβ in contralateral brain tissue.
Results:
1, Model Evaluation
The TTC staining found that there was typical dyeing missing in local ischemic brain tissue, appear white, in line with the disposable area of the middle cerebral artery,and the infarction was relatively stable before and after the optic chiasm. HE staining were observed that, staining shallow in the central farction area, decrease of the cells number, nuclear condensation, smaller and irregular of the nucleolus; interstitial edema; endothelial cell swelling; a large number of grid-like vacuole.
2, The influence of acupuncture treatment to inflammatory response signals in serum: The inflammatory response signals ACTH, IL-1β, IL-6, TNF-α, Hsp70, in cerebral ischemia and reperfusion model rats' serum, showed typical bimodal curve mode in the process of damage development, and the trend were similar. ACTH reached the first peak at12h, and reached the second peak at48h; IL-1β, IL-6, TNF-α, Hsp70reached the first peak at24h, an second peak-to-peak at72-96h. ACTH, IL-6, TNF-α, Hsp70of sham-operated group showed similar bimodal expression trend, IL-1βof sham-operated group was higher than physiological levels only at72h.
Electro-acupuncture treatment on Baihui, Yintang and Renzhong could significantly lowered the the first peak'expression levels of inflammatory response signals ACTH, IL-1β, IL-6, TNF-α, and Hsp70in peripheral serum; could significantly lowered the second peak' expression levels of ACTH, IL-1β, TNF-α; could increase the second peak' expression level of IL-6.
3, The influence of acupuncture treatment to inflammatory response signals in the brain tissue:
The inflammatory response signals in cerebral ischemia and reperfusion model rats' local brain tissue, IL-6, IL-1β, MMP-9, ICAM-1, MCP-1, TGFβshowed typical bimodal curve mode in the process of damage development, and the trend were similar, reached the first peak at12-24h; IL-6reached the second peak at96h; IL-1(3, MMP-9reached the second peak at48h; ICAM-1, MCP-1, TGF(3reached the second peak at72h; TNFαcontinued to increase at12h, reached rhe first peak at24h, maintain a high level to72h. Sham operation group showed similar expression trends.
The inflammatory response signals in cerebral ischemia and reperfusion model rats" contralateral brain tissue, IL-6, IL-1β, TNFα, ICAM-1, MCP-1, TGFp showed typical bimodal curve mode in the process of damage development, to reached the first peak at12-24h; IL-6reached the second peak at96h; IL-1βcontinued to increase from48h, reached the highest value atl44h; TNFareached the first peak at48h; ICAM-1, MCP-1, TGFβreached the second peak at72h; MMP-9reached the first peak at48h, continued to increase from72h, reached the highest value at144h. Sham operation group showed similar expression trends. Electro-acupuncture treatment on Baihui, Yintang and Renzhong had significantly influences on inflammatory response signals in bilateral brain tissue. For the ipsilatcral brain tissue, the expression levels of IL-6, IL-1β, TNFα, MMP-9, MCP-1, TGFβ were lowered; for the contralateral brain tissue, the expression levels of IL-6, MMP-9, MCP-1. TGFβwere lowered; in contralateral brain tissue, the expression levels of IL-6, MMP-9were raises in the acute damage phase at48h; the expression of ICAM-1showed a rising trend before72h in the ipsilateral brain tissue, and before48h in the contralateral brain tissue.
After acupuncture treatment, the second peak' expression of IL-6, ICAM-1in Ipsilateral brain tissue were in early appearance; the second peak' expression of IL-6, IL-1β, MMP-9, ICAM-1, MCP-1TGFβ in contralateral brain tissue were in early appearance.
4, The influence of different acupoints to inflammatory response signals in the local brain tissue: Electro-acupuncture treatment on Baihui, Zusanli, contrast the points Baihui, Yintang and Renzhong, could significantly lower the inflammatory response signals'expression levels in the ipsilateral brain tissue in the rat model, including IL-6, TNF-α, MCP-1, TGFβ, could show the second peak of IL-6, TGFβ in advance.
Conclusions:
1, The brain ischemia reperfusion rat model was stable; cerebral ischemia-reperfusion injury activated the inflammatory/immune-related stress-damage-repair signal chain in body part and the whole, including peripheral serum, bilateral brain tissue; this model was copied successfully.
2, Electro-acupuncture treatment on Baihui, Yintang and Renzhong played the network regulation on stress-damage-repair signal chain, had the overall impact to the inflammatory response signals in he peripheral serum and bilateral brain tissue, including stress signal, endogenous danger signal, start and regulate signals, damage effect signals contact signals, and repair signal; electro-acupuncture may reduce ischemia-reperfusion injury through inhibiting excessive stress, reducing the inflammatory injury, promoting inter-cell contact, starting the repair function in advance, adjusting the healthside brain tissue to compensatory.3, Electro-acupuncture treatment on Baihui, Zusanli, contrast the points Baihui, Yintang and Renzhong, had better influences in regulating the body's inflammatory stress response to injury, reducing the inflammatory response and inflammatory, activating repair function; combined use of acupoints on the head and body may be better in reducing inflammatory injury than using the head acupuncture points only.
引文
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