[Effect of acupuncture on HIF-1α/NLRP3 inflammatory signaling pathway in the rats with cerebral ischemia-reperfusion injury].

OBJECTIVE: To observe the effects of Xingnao Kaiqiao (regaining consciousness and opening orifices) acupuncture therapy on the expression of hypoxia-inducible factor 1α (HIF-1α) and Nod-like receptor protein 3 (NLRP3) in cerebral ischemia-reperfusion rats, and to explore the mechanism of acupuncture against cerebral ischemia-reperfusion injury. METHODS: Seventy-two male SD rats were randomly divided into a sham-operation group, a model group, an acupuncture group and a non-point acupuncture group, with 18 rats in each one. Using modified Longa thread embolization method, the rat model of acute focal cerebral ischemia was prepared; and after 2 h ischemia, the reperfusion was performed to prepared the model of cerebral ischemia-reperfusion. Immediately after reperfusion, Xingnao Kaiqiao acupuncture method was applied to bilateral "Neiguan" (PC 6) and "Shuigou" (GV 26) in the acupuncture group, while in the non-point acupuncture group, acupuncture was delivered at non-points and all of the needles were retained for 30 min in these two groups. The samples were collected 24 h after reperfusion in the rats of each group. Zea-Longa neurological deficit score was used to evaluate the degree of cerebral neurological impairment, TTC staining was adopted to observe the volume percentage of cerebral infarction, HE staining was provided to observe the morphological changes of brain, and Western blot was applied for detecting the expression of HIF-1α and NLRP3 proteins in the cerebral cortex on the right side. RESULTS: Compared with the sham-operation group, neurological deficit score and volume percentage of cerebral infarction were increased in the model group (P<0.01), and HIF-1α and NLRP3 protein expression was elevated (P<0.01). Compared with the model group, neurological deficit score and volume percentage of cerebral infarction were decreased (P<0.01), and HIF-1α and NLRP3 protein expression was lower (P<0.01) in the acupuncture group. There was no significant difference in above indexes in the non-point acupuncture group compared with the model group (P>0.05). Compared with the sham-operation group, the brain tissue of the rats in the model group and the non-point acupuncture group was loose and edema, and the nuclei were shriveled. The brain tissue morphology in the acupuncture group was similar to that of the sham-operation group. CONCLUSION: Acupuncture can alleviate cerebral ischemia-reperfusion injury, and its mechanism may be related to the regulation of HIF-1α/NLRP3 signaling pathway to attenuate inflammatory response.

[Acupuncture regulates autophagic flux to antagonize cerebral ischemic injury in rats].

OBJECTIVE: To observe the effect of acupuncture on the cerebral infarct volume and expressions of Beclin1, microtubule-associated protein 1 light chain 3 (LC3) and p62 proteins related to cell autophagy in rats with cerebral ischemia (CI), so as to explore its mechanisms underlying improvement of CI injury. METHODS: Male SD rats were randomized into 3 groups: sham operation, model and acupuncture which were further divided into 4 subgroups according to different ischemia time-points: 3, 6, 12 and 24 h (n=7 in each subgroup). The CI model was established by occlusion of the middle cerebral artery (MCAO) with surgical suture-embolus. For rats of the acupuncture group, acupuncture was applied to "Shuigou" (GV26) and bilateral "Neiguan" (PC6), and twirled for 1-3 min every time, 10 times altogether, and kept for 30 min. The neurological deficit score accoding to Longa's method was used for assessing the neurological function. The CI volume was measured after 2, 3, 5-triphenyltetrazolium chloride staining. The expression levels of autophagy-related proteins Beclin1，LC3 and p62 in the brain tissue were detected using Western blot. RESULTS: Compared with those of the sham operation group，the neurological deficit scores at 2, 3, 6， 12 and 24 h after CI, and the infarct volumes, the expression levels of Beclin1 and the ratios of LC3-Ⅱ/LC3-Ⅰ at 3, 6, 12 and 24 h were considerably increased (P<0.01, P<0.05), and the expression levels of p62 at 3, 6, 12 and 24 h were significantly decreased (P<0.01) in the model group. Relevant to the model group, acupuncture stimulation of GV26 and PC6 induced an obvious decrease in the neurological deficit scores at 6, 12 and 24 h, CI volumes at 3, 6, 12 and 24 h, and the expression levels of Beclin1 and the ratios of LC3-Ⅱ/LC3-Ⅰ both at 6 and 12 h (P<0.01, P<0.05), and an evident increase in the expression levels of p62 at 6, 12 and 24 h after CI (P<0.05, P<0.01). CONCLUSION: Acupuncture stimulation of GV26 and PC6 can reduce the CI volume and improve neurological function in CI rats， which may be related to its efficacy in down-regulating the expression of Beclin1 and the ratio of LC3-Ⅱ/LC3-Ⅰ, and up-regulating the expression of p62 in the ischemic brain tissue， thereby improving autophagy flux.

[Acupuncture intervention prolongates thrombolysis time window by regulating cerebral cortex angiogenesis-related genes and proteins in cerebral infarction rats].

OBJECTIVE: To observe the effect of acupuncture on the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and endostatin (Endostatin, ES) mRNAs and proteins (angiogenesis related factors) in the ischemic penumbra region in rats with cerebral infarction (CI), so as to explore its underlying mechanisms in prolonging the time window of thrombolysis therapy for CI. METHODS: A total of 48 male SD rats were randomly divided into sham operation, model, medication (6 h thrombolysis) and acupuncture (Acupunct)+medication groups (n=12 in each group). The CI model was established using modified auto-thrombus method. Six hours after thrombolysis, recombinant human tissue plasminogen activator (rt-PA,10 mg/kg) was given to rats of the thrombolysis group through tail vein. Acupuncture was applied at "Shuigou"(CV26) and bilateral "Neiguan" (PC6) 2 h after successful modeling, and the needles were retained for 30 minutes. Cerebral blood flow (CBF) was monitored during modeling in each group, and the neurological deficit score (0-7 points) was given 2 h and 24 h after successful modeling according to Bederson's methods. The cerebral infarction volume was observed after triphenyltetrazole chloride (TTC) staining. The protein and mRNA expression levels of VEGF, bFGF and ES in the ischemic penumbra region of the right cerebral cortex were detected by Western blot and real-time PCR, separately. RESULTS: The neurological deficit score at both 2 h and 24 h after modeling, percentage of cerebral infarction volume, and the expression levels of VEGF, bFGF and ES proteins and mRNAs in the model group were significantly higher than those of the sham operation group (P<0.01, P < 0.05). Compared with the model group, the neurological deficit score 24 h (not at 2 h) after modeling and percentage of cerebral infarction volume, and the expression levels of ES protein and mRNA in the Acupunct+medication group (not in the medication group) were notably lower (P<0.05, P<0.01), while the expression levels of VEGF and bFGF proteins and mRNAs in the Acupunct +medication group (not in the medication group) were considerably higher (P<0.01, P<0.05). No significant differences were found between medication and model groups in the CI percentage, VEGF, bFGF and ES proteins and mRNAs (P>0.05). The therapeutic effect of Acupunct +medication group was significantly superior to that of medication in lowering neurological deficit score, percentage of CI volume and expression of ES protein and mRNA and in up-regulating the expression of VEGF and bFGF proteins and mRNAs (P<0.05, P<0.01). CONCLUSION: Acupuncture and timely intervention can prolong the time window of thrombolysis in CI rats, which may be related to its function in up-regulating the expression of VEGF and bFGF mRNAs and proteins and in down-regulating the expression of ES mRNA and protein in ischemic cerebral cortex.