Electroacupuncture Reduces Cerebral Hemorrhage Injury in Rats by Improving Cerebral Iron Metabolism.

Objective: To study the effects of electroacupuncture at Baihui and Dazhui points on the expression of hepcidin (Hepc), transferrin (Tf), transferrin receptor (TfR), and ferritin (Ft) in rats with cerebral hemorrhage to provide a theoretical basis for the treatment of cerebral hemorrhage with acupuncture. Method: The model of cerebral hemorrhage in rats was established by autologous blood injection method and treated by electroacupuncture (EA) at the acupoints of Baihui and Dazhui. Hepc siRNA was injected into the lateral ventricle 30 min before model preparation to produce the cerebral hemorrhage model. The modified neurological severity score (mNSS) was used to assess the neurological function, and the total iron content in brain tissue was determined using atomic absorption spectrometry; the expression of Hepc, Ft, Tf, and TfR in perihematoma tissue was detected using immunohistochemistry; the interference efficiency of Hepc siRNA was detected using western blot and reverse transcription polymerase chain reaction (RT-PCR). Results: The degree of neurological deficit showed a downward trend at 3 days, 7 days, and 14 days, and electroacupuncture significantly reduced the neurological deficit score at each time point (P < 0.01). Regarding total iron content in brain tissue, on the 3rd day, the 7th day, and the 14th day, the iron content of the hematoma tissue after intracerebral hemorrhage was reduced by electroacupuncture (P < 0.01). Regarding immunohistochemical results. Hepc, Ft, Tf, and TfR protein expressions on day 14 were significantly higher after cerebral hemorrhage (P < 0.01). After electroacupuncture, the expression of Hepc, Ft, Tf, and TfR protein was significantly reduced (P < 0.01). Western blot and RT-PCR revealed that the interference efficiency of Hepc siRNA was statistically significant (P < 0.01). Conclusion: Electroacupuncture can reduce neurological severity scores in rats with cerebral hemorrhage and may exert cerebral protective effects by reducing Hepc protein and gene expression; lowering Ft, Tf, and TfR protein expression; and promoting iron metabolism in the brain of rats with cerebral hemorrhage.

Electroacupuncture at GV20­GB7 regulates mitophagy to protect against neurological deficits following intracerebral hemorrhage via inhibition of apoptosis.

The acupuncture penetrating line of Baihui (GV20) to Qubin (GB7) spans the parietal, frontal and temporal lobes. The present study aimed to elucidate the mechanism by which electroacupuncture (EA) at GV20­GB7 regulates mitophagy in intracerebral hemorrhage (ICH) and whether it serves a neuroprotective role. A whole blood­induced ICH model was used. Mitophagy­regulating proteins, including BCL/adenovirus E1B 19 kDa­interacting protein 3 (BNIP3), PTEN­induced putative kinase 1 (PINK1), Parkin and apoptosis­associated proteins were detected by western blotting; autophagy following ICH was evaluated by immunofluorescent techniques; morphological characteristics of mitophagy were observed using transmission electron microscopy; and TUNEL assay was performed to determine the number of apoptotic cells. Immunohistochemistry was used to detect p53 expression. The protective role of EA (GV20­GB7) via enhanced mitophagy and suppressed apoptosis in ICH was further confirmed by decreased modified neurological severity score. The results showed that EA (GV20­GB7) treatment upregulated mitochondrial autophagy following ICH and inhibited apoptotic cell death. The mechanism underlying EA (GV20­GB7) treatment may involve inhibition of p53, an overlapping protein of autophagy and apoptosis. EA (GV20­GB7) treatment decreased neurobehavioral deficits following ICH but pretreatment with 3­methyladenine counteracted the beneficial effects of EA (GV20­GB7) treatment. In conclusion, EA (GV20­GB7) improved recovery from ICH by regulating the balance between mitophagy and apoptosis.

Acupuncture Ameliorates Neuronal Cell Death, Inflammation, and Ferroptosis and Downregulated miR-23a-3p After Intracerebral Hemorrhage in Rats.

Baihui-penetrating-Qubin acupuncture is frequently used to treat intracerebral hemorrhage (ICH) in China. Acupuncture affects multiple microRNAs in diseases. MicroRNA-23a-3p (miR-23a-3p) has been demonstrated to be up-regulated in ICH patients. Herein, the effect of Baihui-penetrating-Qubin acupuncture on miR-23a-3p expression after ICH and the role of miR-23a-3p in ICH were discussed. A rat model of ICH was induced by infusing autologous blood into caudate nucleus. Acupuncture was performed after ICH once a day for 30 min. After 3 consecutive days of acupuncture, the neurobehavioral function, brain edema, neuronal cell death, inflammation, ferroptosis, nuclear factor E2-like 2 (NFE2L2) signaling and miR-23a-3p levels in brain tissues were analyzed. Additionally, antagomiR-23a-3p was injected into rats 3 days prior to ICH modeling to analyze the function of miR-23a-3p in neuronal cell death, inflammation, ferroptosis, and NFE2L2 signaling. Acupuncture relieved the ICH-induced neurological function deficits, increases in brain water content and Fluoro-Jade B (FJB)-positive cells and release of proinflammatory cytokines. Acupuncture also alleviated ferroptosis and decreased miR-23a-3p expression, as evidenced by the increased NFE2L2 nuclear translocation and expressions of heme oxygenase-1 and glutathione peroxidase 4 and the decreased iron and malondialdehyde contents and reactive oxygen species accumulation. Additionally, antagomiR-23a-3p inhibited the ICH-induced increases in FJB-positive cells, release of proinflammatory cytokines, ferroptosis, and promoted NFE2L2 activation. Notably, the binding site of miR-23a-3p existed in NFE2L2. Taken together, acupuncture may alleviate the neuronal cell death, inflammation, and ferroptosis after ICH by down-regulating miR-23a-3p. This study provides a potential mechanism underlying the Baihui-penetrating-Qubin acupuncture improving the early injury after ICH.

Scalp Acupuncture Attenuates Brain Damage After Intracerebral Hemorrhage Through Enhanced Mitophagy and Reduced Apoptosis in Rats.

To study the effect of scalp acupuncture (SA) on the mitophagy signaling pathway in the caudate nucleus of Sprague-Dawley rats following intracerebral hemorrhage (ICH). An ICH model was established by injecting autologous arterial blood into the caudate nucleus in 200 male Sprague-Dawley rats, which were divided into five groups: sham, ICH, 3-methyladenine group (3-MA, 30 mg/kg), SA, and SA+3-MA. Animals were analyzed at 6 and 24 h as well as at 3 and 7 days. Composite neurological scale score was significantly higher in the SA group than in the ICH group. Transmission electron microscopy showed less structural damage and more autophagic vacuoles within brain in the SA group than in the ICH group. SA group showed higher levels of Beclin1, Parkin, PINK1, NIX protein, and a lower level of Caspase-9 in brain tissue. These animals consequently showed less neural cell apoptosis. Compared with the SA group, however, the neural function score and levels of mitophagy protein in the SA+3-MA group were decreased, neural cell apoptosis was increased with more severe structural damage, which suggested that 3-MA may antagonize the protective effect of SA on brain in rats with ICH. SA may mitigate the neurologic impairment after ICH by enhancing mitophagy and reducing apoptosis.

Acupuncture promotes functional recovery after cerebral hemorrhage by upregulating neurotrophic factor expression.

Acupuncture is widely used in the treatment of cerebral hemorrhage, and it improves outcomes in experimental animal models and patients. However, the mechanisms underlying the effectiveness of acupuncture treatment for cerebral hemorrhage are still unclear. In this study, a model of intracerebral hemorrhage was produced by injecting 50 µL autologous blood into the caudate nucleus in Wistar rats. Acupuncture at Baihui (DU20) and Qubin (GB7) acupoints was performed at a depth of 1.0 inch, 12 hours after blood injection, once every 24 hours. The needle was rotated at 200 r/min for 5 minutes, For each 30-minute session, needling at 200 r/min was performed for three sessions, each lasting 5 minutes. For the positive control group, at 6 hours, and 1, 2, 3 and 7 days after induction of hemorrhage, the rats were intraperitoneally injected with 1 mL aniracetam (0.75 mg/mL), three times a day. The Bederson behavioral test was used to assess palsy in the contralateral limbs. Western blot assay was used to examine the expression levels of Nestin and basic fibroblast growth factor in the basal ganglia. Immunohistochemistry was performed to count the number of Nestin- and glial cell line-derived neurotrophic factor-positive cells in the basal ganglia. Acupuncture effectively reduced hemorrhage and brain edema, elevated the expression levels of Nestin and basic fibroblast growth factor in the basal ganglia, and increased the number of Nestin- and glial cell line-derived neurotrophic factor-positive cells in the basal ganglia. Together, these findings suggest that acupuncture promotes functional recovery after cerebral hemorrhage by increasing the expression of neurotrophic factors. The study was approved by the Committee for Experimental Animals of Heilongjiang Medical Laboratory Animal Center (approval No. 2017061001) on June 10, 2017.

Acupuncture through Baihui (DU20) to Qubin (GB7) mitigates neurological impairment after intracerebral hemorrhage.

Inflammation plays an important role in nerve defects caused by intracerebral hemorrhage. Repairing brain damage by inhibiting the macrophage-inducible C-type lectin/spleen tyrosine kinase (Mincle/Syk) signaling pathway is a potential new target for treating cerebral hemorrhage. In this study, we aimed to determine whether acupuncture through Baihui (DU20) to Qubin (GB7) is an effective treatment for intracerebral hemorrhage through the Mincle/Syk signaling pathway. An intracerebral hemorrhage rat model was established by autologous blood infusion into the caudate nucleus. Acupuncture through Baihui to Qubin was performed for 30 minutes, once every 12 hours, for a total of three times. Piceatannol (34.62 mg/kg), a Syk inhibitor, was intraperitoneally injected as a control. Modified neurological severity score was used to assess neurological function. Brain water content was measured. Immunohistochemistry and western blot assay were used to detect immunoreactivity and protein expression levels of Mincle, Syk, and CARD9. Real-time polymerase chain reaction was used to determine interleukin-1ß mRNA levels. Hematoxylin-eosin staining was performed to observe histopathological changes. Our results showed that acupuncture through Baihui to Qubin remarkably improved neurological function and brain water content, and inhibited immunoreactivity and expression of Mincle, Syk, CARD9, and interkeukin-1ß. Moreover, this effect was similar to piceatannol. These findings suggest that acupuncture through Baihui to Qubin can improve neurological impairment after cerebral hemorrhage by inhibiting the Mincle/Syk signaling pathway.

[Penetrative Needling from "Baihui" (GV 20) to "Qubin" (GB 7) Antagonizes Inflammatory Injury in Rats with Cerebral Hemorrhage].

OBJECTIVE: To observe the effect of penetrative needling of "Baihui" (GV 20) to "Qubin" (GB 7) on neurologic functions and expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and toll-like receptor 4 (TLR-4, involving in inflammatory reactions) in the tissue around the local cerebral hematoma in rats with intracerebral hemorrhage (ICH), so as to provide evidence for clinical treatment of ICH. METHODS: Fifty-four male Wistar rats were randomly divided into sham control, mo-del and acupuncture groups, and then further divided into three time-point subgroups(1,3,7 days after modeling, n=6/subgroup). The ICH model was established by injection of the rat's autoblood (50 µL) into the putaman region (P:0.2 mm, R:3.5 mm) in a stereotaxic apparatus and confirmed by Berderson's neurologic examination grading system (0-3 points). The neurologic function was assessed by using Longa's scoring (5-points) and footfault asymmetry testing[footfault index=(contra faults-ipsi faults)/total steps in 2 min]. For penetrative needling, an acupuncture needle was inserted into GV 20 and controlled to advance to GB 7 on the affected side and retained for 30 min, once daily. The expression of TNF-α, IL-6 and TLR-4 in the cerebral tissue around the putaman was detected by immunohistochemistry. RESULTS: After penetrative needling stimulation, the increased Longa's score and footfault asymmetry score in ICH rats were significantly decreased on day 1, 3 and 7 after modeling (P<0.01), suggesting an improvement of neurologic function after the treatment. Immunohistochemical staining outcomes of the cerebral tissue surrounding the autoblood injection site showed that the expression levels of TNF-α, IL-6 and TLR-4 proteins on day 1, 3 and 7 were considerably higher in the model group than in the control group (P<0.01), and markedly lower in the acupuncture group than in the model group (P<0.01), suggesting a suppression of the proinflammatory factors and TLR-4 levels around the locus of the brain after needling intervention. A positive correlation existed between the expression levels of TLR-4 and IL-6/TNF-α. CONCLUSIONS: Penetrative needling stimulation of GV 20 to GB 7 can reduce the levels of proinflammatory factors TNF-α and IL-6, and TLR-4 in the ICH tissues in rats with cerebral hemorrhage, which may contribute to its effect in improving neurological function.

Acupuncture inhibits Notch1 and Hes1 protein expression in the basal ganglia of rats with cerebral hemorrhage.

Notch pathway activation maintains neural stem cells in a proliferating state and increases nerve repair capacity. To date, studies have rarely focused on changes or damage to signal transduction pathways during cerebral hemorrhage. Here, we examined the effect of acupuncture in a rat model of cerebral hemorrhage. We examined four groups: in the control group, rats received no treatment. In the model group, cerebral hemorrhage models were established by infusing non-heparinized blood into the brain. In the acupuncture group, modeled rats had Baihui (DU20) and Qubin (GB7) acupoints treated once a day for 30 minutes. In the DAPT group, modeled rats had 0.15 µg/mL DAPT solution (10 mL) infused into the brain. Immunohistochemistry and western blot results showed that acupuncture effectively inhibits Notch1 and Hes1 protein expression in rat basal ganglia. These inhibitory effects were identical to DAPT, a Notch signaling pathway inhibitor. Our results suggest that acupuncture has a neuroprotective effect on cerebral hemorrhage by inhibiting Notch-Hes signaling pathway transduction in rat basal ganglia after cerebral hemorrhage.