针刺百会、曲鬓穴对急性期脑出血大鼠的影响及其作用机制研究

Interventional Effect and Mechanism of Acupuncture at Baihui(GV 20)and Qubin(GB 7)Acupoints on Rats with Acute Cerebral Hemorrhage

目的 观察针刺百会、曲鬓穴对急性期脑出血(ICH)大鼠白细胞分化抗原36(CD36)、Toll样受体4(TLR4)表达的影响,探讨针刺治疗脑出血的作用机制。方法 选择144只Wistar雄性大鼠,采用随机数字表法分为假手术组、模型组、针刺组、抑制剂组4组,每组36只,每组按1、3、7 d时间点再分为3个亚组,每组12只。采用立体定位自体血注入法建立ICH大鼠模型。模型组仅接受ICH模型制备,不进行任何治疗;假手术组接受类似模型组各项手术操作,但不进行注血制作;抑制剂组造模后6 h,腹腔注射TLR4抑制剂TAK242,3 mg/kg,1次/d,连续5 d;造模12 h后,针刺组各亚组开始接受针刺治疗,穴位选择百会穴(顶骨正中)、右侧曲鬓穴,采用透刺方法,进针深度20 mm,以100 r/min小幅度捻转,持续捻转2 min,每间隔5 min捻针1次,共留针30 min,期间捻转3次,1次/d,针刺组各亚组分别治疗1、3、7 d。分别于治疗后第1、3、7天,采用改良神经功能缺损评分(mNSS)评估大鼠神经功能;检测脑组织血肿体积;采用Western blot法检测脑组织CD36、TLR4蛋白表达水平;采用免疫荧光法观察CD36、TLR4在星形胶质细胞中的表达。结果 (1) mNSS评分:与假手术组同一时间点比较,模型组、针刺组、抑制剂组治疗后1、3、7 d mNSS评分均明显升高(P<0.05);与模型组同一时间点比较,针刺组、抑制剂组治疗后1、3、7 d mNSS评分均明显降低(P<0.05);与针刺组同一时间点比较,抑制剂组治疗后1 d mNSS评分明显降低(P<0.05)。(2)血肿体积:与模型组同一时间点比较,针刺组、抑制剂组治疗后3、7 d脑血肿体积均明显降低,抑制剂组治疗后1、3、7 d脑血肿体积明显降低(P<0.05);与针刺组同一时间点比较,抑制剂组治疗后1、3、7 d脑血肿体积明显降低(P<0.05)。(3) CD36、TLR4蛋白表达水平:与假手术组同一时间点比较,模型组、针刺组、抑制剂组治疗后1、3、7 d CD36、TLR4蛋白表达水平均明显升高(P<0.05);与模型组同一时间点比较,针刺组、抑制剂组治疗后1、3、7 d CD36蛋白表达水平均明显升高(P<0.05),针刺组、抑制剂组治疗后3、7 d TLR4蛋白表达水平均明显降低(P<0.05);与针刺组比较,抑制剂组治疗后1、3、7 d CD36蛋白表达水平均明显升高,TLR4蛋白表达水平均明显降低(P<0.05)。(4) CD36、TLR4在GFAP中的表达水平:假手术组大鼠脑组织内可见少量CD36、TLR4表达。与假手术组同一时间点比较,模型组治疗后1、3、7 d CD36、TLR4在GFAP表达均明显增加(P<0.05);与模型组同一时间点比较,抑制剂组、针刺组治疗后1、3、7 d CD36在GFAP表达明显增加,TLR4在GFAP表达明显降低(P<0.05)。结论 针刺百会、曲鬓穴可以改善急性期ICH大鼠神经功能,减轻脑出血血肿体积,可能与促进CD36蛋白表达、抑制TLR4蛋白表达有关。

Objective To investigate the interventional effect of acupuncture at Baihui(GV 20)and Qubin(GB 7)acupoints on the expression of recombinant cluster of differentiation 36(CD36)and Toll-like receptor 4(TLR4)in rats with acute cerebral hemorrhage(ICH),and to explore the mechanism of acupuncture treatment for ICH.Methods A total of 144 male Wistar rats were randomly divided into sham operation group,model group,acupuncture group and inhibitor group,with 36 cases in each group,and then each group was divided into three subgroups at the 1st,3rd and 7th day,with 12 cases in each subgroup.The stereotaxic autolo‐gous blood injection method was used to establish a rat model of ICH.The model group only received ICH model preparation with‐out any treatment.The sham operation group received various operations similar to the model group,but without blood injection.The inhibitor group received an intraperitoneal injection of the TLR4 inhibitor TAK242 at 6 hours after modeling,and the dose was 3 mg/kg,once daily for 5 days.At 12 hours after model establishment,all subgroups of the acupuncture group received penetrative needling at Baihui(GV 20)and Qubin(GV 25)acupoints.The needle was retained at a depth of 20 mm and twisted at a frequency of 100 r/min for three sessions of 2 min each at an interval of 5 min during the 30-min needle retention period.Acupuncture was given once a day and lasted for 1,3,and 7 days in the three acupuncture subgroups,respectively.At the 1st,3rd and 7th day after treat‐ment,modified neurological severity score(mNSS)was used to assess neurological function.The volume of hematoma in brain tis‐sue was detected.Western blot was used to detect the protein expression level of CD36 and TLR4 in brain tissue.Immunofluores‐cence method was used to detect the expression of CD36 and TLR4 in astrocytes.Results(1)mNSS score:compared with the sham operation group at the same time,mNSS score of the model group,the acupuncture group and the inhibitor group at 1,3,7 days after treatment increased significantly(P<0.05);compared with the model group at the same time,mNSS score of the acupuncture group and the inhibitor group at 1,3,7 days after treatment decreased significantly(P<0.05);compared with the acupuncture group at the same time,mNSS score in the inhibitor group at 1 day after treatment decreased significantly(P<0.05).(2)Hematoma volume:compared with the model group at the same time,hematoma volume in the acupuncture group and the inhibitor group at 3,7 days after treatment decreased significantly,and hematoma volume in the inhibitor group at 1,3,7 days after treatment decreased signifi‐cantly(P<0.05);compared with the acupuncture group at the same time,hematoma volume in the inhibitor group decreased signifi‐cantly at 1,3,7 days after treatment decreased significantly(P<0.05).(3)Protein expression level of CD36 and TLR4:compared with the sham operation group at the same time,the protein expression level of CD36 and TLR4 in the model group,the acupuncture group and the inhibitor group at 1,3,7 days after treatment increased significantly(P<0.05);compared with the model group at the same time,the expression level of CD36 protein in the acupuncture group and the inhibitor group at 1,3,7 days after treatment in‐creased significantly(P<0.05),and the expression level of TLR4 protein in the acupuncture group and the inhibitor group at 3,7 days after treatment decreased significantly(P<0.05);compared with the acupuncture group,the expression level of CD36 protein in the inhibitor group at 1,3,7 days after treatment increased significantly,and the expression level of TLR4 protein decreased signifi‐cantly(P<0.05).(4)Expression level of CD36 and TLR4 in GFAP:there was a small amount of CD36 and TLR4 in the brain tissue of rats in the sham operation group.Compared with the sham operation group at the same time,the expression of CD36 and TLR4 in GFAP in the model group at 1,3,7 days after treatment increased significantly(P<0.05);compared with the model group at the same time,the expression of CD36 in GFAP of the inhibitor group and the acupuncture group at 1,3,7 days after treatment in‐creased significantly,while the expression of TLR4 in GFAP decreased significantly(P<0.05).Conclusion Acupuncture at Baihui(GV 20)and Qubin(GB 7)acupoints can improve neurological function of rats with acute ICH,alleviate hematoma volume,which may be related to promoting the expression of CD36 protein and inhibiting the expression of TLR4 protein.