Suture-occluded Method Research Articles

Acupuncture modulates the AMPK/PGC-1 signaling pathway to facilitate mitochondrial biogenesis and neural recovery in ischemic stroke rats.

The main objective of this study was to investigate the role and mechanism of acupuncture on anti-nerve injury in the acute phase by regulating mitochondrial energy metabolism via monophosphate-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) axis in rat ischemic stroke. Middle cerebral artery occlusion (MCAO) was established by middle cerebral artery occlusion/reperfusion. One-week of acupuncture was performed during the acute phase of ischemic stroke. The neurological function and brain tissue integrity were evaluated. Mitochondrial function (intracellular ATP level and the activity of mitochondrial respiratory chain complex I) and the level of NADH oxidase (NOX) were detected by enzymatic chemistry. Next, the potential molecular mechanisms were explored by western blotting, fluorescence quantitative PCR and immunohistochemistry method. (1) Acupuncture treatment for MCAO/R rats showed a significant improvement in the infarcted tissue accompanied by functional recovery in Zea-Longa score and balance beam score outcomes, motor function performances. (2) Acupuncture increased the levels of ATP and mitochondrial respiratory chain complex I, decreased the NOX levels in cerebral ischemia established by suture-occluded method. (3) Acupuncture reduced the necrosis dissolution of neuronal cells and meningeal edema, while promoting angiogenesis. (4) Quantitative immunohistochemical staining results showed acupuncture can increase the expression of AMPK, p-AMPK and the mitochondrial transcription factor PGC-1α, NRF2, TFAM and uncoupling protein 2 (UCP2). Meanwhile, acupuncture treatment up-regulated the expression of the corresponding protein. (5) Subsequently, acupuncture enhanced AMPK phosphorylation as well as the expression of PGC-1α, NRF2, TFAM and UCP2, implicated in mitochondrial synthesis and cellular apoptosis. (6) Finally, injections of AMPK antagonists and activators confirmed AMPK as a therapeutic target for the anti-nerve damage effects of acupuncture. Acupuncture intervention relieved ischemic stroke progression in MCAO rats by promoting energy metabolism and mitochondrial biogenesis in the brain and alleviating neuronal apoptosis, which was mediated by eliciting AMPK/PGC-1α axis, among them AMPK is a therapeutic target.

Pharmacodynamics and mechanism of Erigeron breviscapus granules in the treatment of ischemic stroke in mice by regulating sphingolipid metabolism based on metabolomics

AimErigeron breviscapus (Vant.) Hand.-Mazz. (EB) granules is the extract preparation of EB, with clear curative effect and unclear mechanism. This study intends to systematically explore the specific mechanism of EB granules in the treatment of IS from the metabolic perspective. MethodsThe model of transient middle cerebral artery occlusion (tMCAO) in mice was established by the suture-occluded method. The therapeutic effect of EB granules on tMCAO mice was evaluated by behavioral evaluation, brain water content determination, 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (HE) staining, and levels of lactate dehydrogenase (LDH) and neuron specific enolase (NSE) in serum. In order to screen differential metabolites, non-targeted metabolomics technology was used to detect the metabolites in serum before and after administration. Univariate statistics, multivariate statistics and bioinformatics were used to analyze the changes of metabolites in serum of tMCAO mice. The possible related mechanism of EB granules in treating IS was screened by pathway enrichment analysis, and the preliminary verification was carried out at animal level by enzyme linked immunosorbent assay (ELISA) and western blot (WB). ResultsEB granules could significantly improve behavior of tMCAO mice, reduce brain water content and cerebral infarction volume, improve morphology of brain tissue, reduce the levels of LDH and NSE in serum. A total of 232 differential metabolites were screened, which were mainly enriched in many biological processes such as sphingolipid metabolism. The differential metabolite S1P and its receptors S1PR1 and S1PR2 in sphingolipid metabolism were verified. The results showed that the level of S1P in brain tissue increased and the protein expression of S1PR1 decreased significantly after modeling, and reversed after administration, but there was no significant difference in the protein expression of S1PR2. ConclusionThe therapeutic effects of EB granules may be related to affecting sphingolipid metabolism through regulating S1P/S1PR1.

Study on the action mechanism of Buyang Huanwu Decoction against ischemic stroke based on S1P/S1PR1/PI3K/Akt signaling pathway

Ethnopharmacological relevanceIschemic stroke is a common and frequent clinical disease. Recent studies have demonstrated that sphingolipid plays an important role in the pathological process of ischemic stroke. PI3K-Akt is a classic protective signaling pathway of cerebral ischemic injury. After acting on the S1P receptor, S1P can activate the downstream PI3K/Akt signaling pathway and play an anti-cerebral ischemia role. Buyang Huanwu Decoction (BHD) is a traditional Chinese medicine formula used to treat ischemic stroke. However, the mechanisms of BHD on ischemic stroke remain unclear based on S1P/S1PR1/PI3K/Akt signaling pathway. Aim of the studyThe present study is intended to investigate the action mechanism of BHD on ischemic stroke based on the S1P/S1PR1/PI3K/Akt signaling pathway from multiple perspectives. Materials and methodsThe BHD lyophilized product was prepared by vacuum freeze-drying method, of which the chemical composition was determined by UPLC-Q-TOF/MS. The mouse permanent middle cerebral artery occlusion (pMCAO) model was established by the suture-occluded method. Male KM mice were randomly divided into seven groups: sham group, model group, FTY720 (positive control) group, BHD group, BHD + W146 (selective S1PR1 inhibitor) group, SEW2871 (selective S1PR1 agonist) group, and Calycosin group. Each group was administered continuously for 14 days and evaluated with modified neurological severity score (mNSS) and cerebral infarct volume on the 1st, 4th, 7th, and 14th days. The SphK1, SphK2, S1PR1, PI3K, Akt, and p-Akt protein in the prefrontal lobe, hippocampus, and striatum was quantified by Western blot and immunohistochemical (IHC) experiment respectively. The qRT-PCR method was employed to evaluate SphK1, SphK2, and S1PR1 mRNA expression in the above tissue. ResultsBHD and Calycosin both effectively improved mNSS scores with smaller infarct volumes. The SphK1 level in the prefrontal lobe, hippocampus, and striatum of mice in the BHD group was significantly lower, and SphK2, PI3K, and p-Akt in the hippocampus and striatum were significantly higher than those in the model group.BHD significantly decreased SphK1 mRNA expression in the prefrontal lobe, hippocampus, and striatum, and significantly up-regulated SphK2 mRNA and S1PR1 mRNA expression. Additionally, SphK1 protein expression levels of the prefrontal lobe, hippocampus, and striatum in the BHD group was significantly lower than model group, and SphK2, S1PR1, PI3K, Akt, and p-Akt protein expressions levels were increased obviously.Furthermore, SEW2871 can increase S1PR1 and Akt expression, and up-regulate SphK2 and S1PR1 mRNA expression. The effect of BHD on the expression of S1P/S1PR1/PI3K/Akt signaling pathway-related proteins and mRNA were weakened by BHD + W146. ConclusionBHD and Calycosin significantly improved the symptoms of neurological deficits in pMCAO mice, reduced the cerebral infarction volume, up-regulated SphK2 and S1PR1 mRNA levels, enhanced SphK2, S1PR1, PI3K, Akt, p-Akt protein expression of the prefrontal lobe, hippocampus and striatum, and down-regulated SphK1 mRNA and protein expression, which may be helpful to clarify the mechanism of BHD through S1P/S1PR1/PI3K/Akt signaling pathway to protect against cerebral ischemic injury.

Protective effects of three kinds of borneol on different brain regions in acute cerebral ischemia/reperfusion model rats

This study compared the ameliorating effects of L-borneol, natural borneol, and synthetic borneol on the injury of different brain regions in the rat model of acute phase of cerebral ischemia/reperfusion(I/R) for the first time, which provides a reference for guiding the rational application of borneol in the early treatment of ischemic stroke and has important academic and application values. Healthy specific pathogen-free(SPF)-grade SD male rats were randomly assigned into 13 groups: a sham-operation group, a model group, a Tween model group, a positive drug(nimodipine) group, and high-, medium-, and low-dose(0.2, 0.1, and 0.05 g·kg~(-1), respectively) groups of L-borneol, natural borneol, and synthetic borneol according to body weight. After 3 days of pre-administration, the rat model of I/R was established by suture-occluded method and confirmed by laser speckle imaging. The corresponding agents in different groups were then administered for 1 day. The body temperature was monitored regularly before pre-administration, days 1, 2, and 3 of pre-administration, 2 h after model awakening, and 1 d after model establishment. Neurological function was evaluated based on Zea-Longa score and modified neurological severity score(mNSS) 2 h and next day after awakening. The rats were anesthetized 30 min after the last administration, and blood was collected from the abdominal aorta. Enzyme-linked immunoassay assay(ELISA) was employed to determine the serum levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-4, and transforming growth factor-beta1(TGF-β1). The brain tissues were stained with triphenyltetrazolium chloride(TTC) for the calculation of cerebral infarction rate, and hematoxylin-eosin(HE) staining was used for observing and semi-quantitatively evaluating the pathological damage in different brain regions. Immunohistochemistry was employed to detect the expression of ionized calcium binding adapter molecule 1(IBA1) in microglia. q-PCR was carried out to determine the mRNA levels of iNOS and arginase 1(Arg1), markers of polarization phenotype M1 and M2 in microglia. Compared with the sham-operation group, the model group and the Tween model group showed significantly elevated body temperature, Zea-Longa score, mNSS, and cerebral infarction rate, severely damaged cortex, hippocampus, and striatum, increased serum levels of IL-6 and TNF-α, and decreased serum levels of IL-4 and TGF-β1. The three borneol products had a tendency to reduce the body temperature of rats 1 day after modeling. Synthetic borneol at the doses of 0.2 and 0.05 g·kg~(-1), as well as L-borneol of 0.1 g·kg~(-1), significantly reduced Zea-Longa score and mNSS. The three borneol products at the dose of 0.2 g·kg~(-1) significantly reduced the cerebral infarction rate. L-borneol at the doses of 0.2 and 0.1 g·kg~(-1) and natural borneol at the dose of 0.1 g·kg~(-1) significantly reduced the pathological damage of the cortex. L-borneol and natural borneol at the dose of 0.1 g·kg~(-1) attenuated the pathological damage of hippocampus, and 0.2 g·kg~(-1) L-borneol attenuated the damage of striatum. The 0.2 g·kg~(-1) L-borneol and the three doses of natural borneol and synthetic borneol significantly reduced the serum level of TNF-α, and the 0.1 g·kg~(-1) synthetic borneol reduced the level of IL-6. L-borneol and synthetic borneol at the dose of 0.2 g·kg~(-1) significantly inhibited the activation of cortical microglia, and 0.2 g·kg~(-1) L-borneol up-regulated the expression of Arg1 and down-regulated the expression level of iNOS. In conclusion, the three borneol products may alleviate inflammation to ameliorate the pathological damage of brain regions of rats in the acute phase of I/R by inhibiting the activation of microglia and promoting the polarization of microglia from M1 type to M2 type. The protective effect on brain followed a trend of L-borneol > synthetic borneol > natural borneol. We suggest L-borneol the first choice for the treatment of I/R in the acute phase.

Advanced Glycation End-Products (AGEs) Promote Endothelial Cell Pyroptosis Under Cerebral Ischemia and Hypoxia via HIF-1α-RAGE-NLRP3.

This work mainly aimed to explore the role and mechanism of advanced glycation end-products (AGEs) in inducing cerebrovascular endothelial cell pyroptosis under oxygen glucose deprivation (OGD) condition. The mouse cerebral microvascular endothelial cells (BMECs and bEnd.3) were used as the objects to construct the OGD model in vitro. Then, cells were pretreated with AGE-modified human serum albumin (AGE-HSA). Thereafter, CCK-8 assay was conducted to detect cell viability, and flow cytometry (FCM) was performed to measure cell pyroptosis level. Meanwhile, the expression of inflammatory factors was detected by enzyme-linked immunosorbent assay (ELISA). The expression of HIF-α, NLRP3, and RAGE was detected by fluorescence staining. The opening status of cell membrane pore was observed under the electron microscope, and the expression levels of FL-GSDMD, NT-GSDMD, and caspase-1 were measured through Western Blot (WB) assay. Moreover, bEnd.3 cells were treated with siRAN-silenced NLRP3 and HIF-α inhibitor, so as to observe the effect of AGEs on cell pyroptosis level. In the mouse model, the middle cerebral artery occlusion (MCAO) model was constructed by the suture-occluded method. After intraperitoneal injection of AGEs, the pathological changes in mouse brain tissues were detected; the expression levels of NLRP3, ZO-1, and CD31 were determined by histochemical staining, and the levels of inflammatory factors and pyroptosis-related proteins were also detected. Under OGD condition, AGEs induced the pyroptosis of bEnd.3 cells, and the cell pyroptosis rate increased, higher than that of the OGD group. Meanwhile, the levels of inflammatory factors were up-regulated; the expression of HIF-α, NLRP3, and RAGE in cells increased; and the levels of NT-GSDMD and caspase-1 were markedly higher than those of the control and OGD groups. siRNA-NLRP3 or HIF-α inhibitor treatment suppressed pyroptosis and reduced the inflammatory factor levels. In mouse experiments, AGE injection aggravated brain injury in the MCAO mouse model, decreased the expression of ZO-1 and CD31, and elevated the levels of NLRP3 and inflammatory factors. Under cerebral ischemia condition, AGEs can induce endothelial cell pyroptosis via HIF-α-RAGE-NLRP3, thereby further aggravating brain injury.

Anti-inflammation mechanism of electro-scalp acupuncture in treatment of ischemic stroke based on IL-12 mediated JAK/STAT signaling pathway

To observe the impact of electro-scalp acupuncture (ESA) on the neural function and inflammatory response of ischemic cortex in the model rats with ischemic stroke and explore the anti-inflammation mechanism of ESA in treatment of ischemic stroke from the perspective of modulating the interleukin 12 (IL-12) mediated JAK (Janus kinase)/STAT (signal transduction and transcription activator) signal pathway. Ninety male SD rats were randomized into a normal group (n =16) and a model preparation group (n=74). In the model preparation group, the model of middle cerebral artery occlusion (MCAO) was duplicated with suture-occlusion method. After modeled successfully, 48 rats with neurological deficit score of 1-3 were divided into a model group, an inhibitor group and an ESA group, 16 rats in each one. In the inhibitor group, IL-12 inhibitor (apilimod, 5 mg/kg) was used via intragastric administration. In the ESA group, the anterior oblique line of vertex-temporal (MS6) was stimulated bilaterally with electric acupuncture, with disperse-dense wave, 2 Hz/100 Hz in frequency, 1 mA in current intensity. The needles were retained for 30 min. The treatment was given once daily and for 7 days in above two intervention groups. Before and after intervention, the neurological deficit score (NDS) and neurobehavioral score (NBS) were assessed in each group. HE staining method was adopted to observe the morphological manifestations of ischemic cortical lesion; the concentrations of IL-12 and IL-12R of the brain tissue in the ischemic cortical lesion were measured by enzyme-linked immunosorbent assay (ELISA); the mRNA expression levels of STAT4 and Tbx21 were detected by real-time PCR technique; and the protein expression of IL-2, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-4 were detected using immunohistochemistry. NDS and NBS in the model group, the inhibitor group and the ESA group were all higher than those in the normal group before intervention (P<0.01). After intervention, NDS and NBS in the model group were higher than the normal group (P<0.01); the two scores were all reduced when compared with those before intervention in the inhibitor group and the ESA group (P<0.01), and lower than those of the model group (P<0.01). NDS in the ESA group was lower than the inhibitor group (P<0.05). In the model group, the cells were shrunk and vacuolated in the ischemic cortical lesion. Many normal cells were visible in the ESA group and the inhibitor group. Compared with the normal group, the concentrations of IL-12 and IL-12R , the mRNA expression levels of STAT4 and Tbx21 and the protein expression levels of IL-2, TNF-α and IFN-γ in brain tissue of ischemic cortical lesion were all increased in the model group (P<0.01), while the protein expression level of IL-4 decreased (P<0.01). The concentrations of IL-12 and IL-12R, the mRNA expression levels of STAT4 and Tbx21 and the protein expression levels of IL-2, TNF-α and IFN-γ were all reduced (P<0.01), while the protein expression level of IL-4 increased (P<0.01) in the ESA group and the inhibitor group when compared with the model group. The concentration of IL-12, the mRNA expression levels of STAT4 and Tbx21 and the protein expression levels of IL-2, TNF-α and IFN-γ in the ESA group were all higher than those of the inhibitor group (P<0.05); while the concentration of IL-12R and the protein expression level of IL-4 were lower than the inhibitor group (P<0.05). Electro-scalp acupuncture may improve the neurological function of the rats with ischemic stroke. The modulation to IL-12 mediated JAK/STAT signaling pathway is the potential molecular mechanism of this therapy for the inflammatory response in ischemic cortical lesion.

Chuanzhi Tongluo Capsules antagonizes cerebral ischemia-reperfusion injury in mice by inhibiting neuroinflammation and oxidative stress

Chuanzhi Tongluo Capsules(CZTL) is effective in activating blood, resolving stasis, replenishing Qi, and dredging collaterals, which has been widely used in clinical treatment of stroke(cerebral infarction) differentiated into the syndrome of wind striking meridian and collateral in the recovery stage characterized by blood stasis and Qi deficiency. However, its modern pharmacological mechanisms of action remain unclear. This study duplicated the middle cerebral artery occlusion and reperfusion(MCAO/R) model in mice using the suture-occluded method to explore the protective effect and mechanism of CZTL on ischemic stroke. The mice were divided into the sham-operation group, model group, and CZTL group. The ones in the CZTL group were gavaged with 0.3 g·kg~(-1)·d~(-1) CZTL for three successive days. One hour after the last intragastric administration, those in the model and CZTL groups were subjected to MCAO/R. After 24 h reperfusion, the effects of CZTL on neurological deficit score, cerebral infarction area, brain edema, and brain histopathology were evaluated. The levels of reactive oxygen species(ROS), malondialdehyde(MDA), interleukin-6(IL-6), interleukin-1β(IL-1β), and tumor necrosis factor-α(TNF-α) and the activity of superoxide dismutase(SOD) in brain tissue homogenate were detected using corresponding assay kits. The expression of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), Toll like receptor 4(TLR4), and phosphorylated nuclear factor-κB P65 subunit(p-NF-κB P65) were assayed by Western blot. The results indicated that CZTL significantly reduced the neurological deficit score, brain edema, and infarct volume, improved the brain histopathology, inhibited the expression of ROS, MDA, IL-6, IL-1β, and TNF-α in the brain tissue, and up-regulated the activity of SOD, down-regulated the expression of pro-apoptotic protein Bax, promoted the expression of anti-apoptotic protein Bcl-2, and suppressed the expression of TLR4 and p-NF-κB P65 phosphorylation of MCAO/R mice. All these have demonstrated that CZTL has a significant protective effect against MCAO/R injury in mice, which may be related to its resistance to neuroinflammation and oxidative stress.

Dedicator of Cytokinesis 2 (DOCK2) Silencing Protects Against Cerebral Ischemia/Reperfusion by Modulating Microglia Polarization via the Activation of the STAT6 Signaling Pathway

Cerebral ischemia/reperfusion is the major pathophysiological process in stroke and could lead to severe and permanent disability. The current study aimed to investigate the effects of dedicator of cytokinesis 2 (DOCK2) on cerebral ischemia/reperfusion-induced cerebral injury. We established a mouse middle cerebral artery occlusion (MCAO) model with suture-occluded method in vivo. Then, BV-2 cells were conducted to oxygen-glucose deprivation and re-oxygenation (OGD/R) in vitro. The results showed that DOCK2 was highly expressed in ischemic brain following MCAO and in BV-2 cells induced by OGD/R. DOCK2 depletion protected against MCAO-induced brain infarcts and neuron degeneration. DOCK2 downregulation improved long-term neurological function, which was assessed by the Morris water-maze test. Moreover, silencing of DOCK2 promoted M2 polarization (anti-inflammation) and repressed M1 polarization (pro-inflammation) of microglia both in vivo and in vitro. Subsequently, we found that the loss of DOCK2 upregulated the expression of p-STAT6. DOCK2 knockdown-induced microglial cell polarization towards M2 phenotype was partly abrogated by the STAT6 inhibitor AS1517499. In conclusion, DOCK2 downregulation protected against cerebral ischemia/reperfusion by modulating microglia polarization via the activation of the STAT6 signaling pathway.

Molecular Mechanism of Electroacupuncture Regulating Cerebral Arterial Contractile Protein in Rats with Cerebral Infarction Based on MLCK Pathway.

To explore the effect of electroacupuncture (EA) intervention on the vasoconstriction of cerebral artery smooth muscle cells after cerebral infarction. Male Wistar rats were randomly divided into 3 groups by a random number table: the model group (n=24), the EA group (n=24), and the normal group (n=6). The model and the EA groups were divided into different time subgroups at 0.5, 1, 3, and 6 h after middle cerebral artery occlusion (MCAO), with 6 rats in each subgroup. MCAO model was established using intraluminal suture occlusion method. The EA group was given EA treatment at acupoint Shuigou (GV 26) instantly after MCAO for 20 min. The contents of cerebrovascular smooth muscle MLCK, the 3 subunits of myosin light chain phosphatase (MLCP) MYPT1, PP1c-δ and M20, as well as myosin-ATPase activity were detected using immunohistochemistry and Western blotting. The overall expression level of the MYPT1 and PP1c-δ in the model group was significantly higher (P<0.01). After EA intervention, the 0.5 h group expression level was close to that of the normal group (P>0.05), and the other subgroups were still significantly higher than the normal group (P<0.01). After EA intervention, the expression level of each subgroup was significantly lower than the corresponding model group. There was a significant difference between the 0.5 and 1 h subgroups (P<0.01), while a difference was also observed between the 3 and 6 h subgroups (P<0.05). The dynamic change rule gradually increased with the prolongation of infarction time within 6 h after infarction. EA intervention can inhibit contraction of cerebral vascular smooth muscle cells and regulate smooth muscle relaxation by regulating MLCK pathway.

MicroRNA-193b-3p reduces oxidative stress and mitochondrial damage in rats with cerebral ischemia-reperfusion injury via the seven in absentia homolog 1/Jun N-terminal kinase pathway

ABSTRACT Ischemic stroke is one of the major causes of death and disability among adults. This study sought to explore the mechanism of microRNA (miR)-193b-3p in rats with cerebral ischemia-reperfusion (I/R) injury. The cerebral I/R injury models of rats were established using the suture-occluded method. The pathological changes were observed, and oxidative stress (OS) and mitochondrial function indexes in rat brain tissue were examined. The levels of miR-193b-3p and seven in absentia homolog 1 (SIAH1) were detected. miR-193b-3p agomir or antagomir was injected into the lateral ventricle of I/R rats to overexpress or inhibit miR-193b-3p expression. The targeting relationship between miR-193b-3p and SIAH1 was verified. The effect of SIAH1 overexpression on brain injury in I/R rats was investigated by injecting the lentivirus vector into the lateral ventricle. The phosphorylation level of Jun N-terminal kinase (JNK) was identified. miR-193b-3p was lowly expressed in I/R rats. Overexpression of miR-193b-3p alleviated the pathological damage of I/R rats and limited the OS and mitochondrial damage. miR-193b-3p targeted SIAH1. Overexpression of SIAH1 partially reversed the protection of miR-193b-3p overexpression against cerebral I/R injury. p-JNK was up-regulated in I/R rats and overexpression of miR-193b-3p inhibited p-JNK. Overall, overexpression of miR-193b-3p targeted SIAH1 to inhibit the activation of the JNK pathway and protect rats against cerebral I/R injury.

Effect of transcutaneous auricular vagus nerve stimulation on the expressions of GFAP and MAP2 in ischemic penumbra of rats with middle cerebral artery ischemia

To observe the effects of transcutaneous auricular vagus nerve stimulation (taVNS) on the motor function and the expression of glial fibrillary acidic protein (GFAP) and microtubule associated protein 2 (MAP2) in cerebral ischemic penumbra of rats with middle cerebral artery occlusion (MCAO) and explore the mechanism of taVNS in the improvement of motor function in MCAO rats. A total of 48 male SD rats were randomized into a sham-operation group, a model group, a transcutaneous auricular non-vagus nerve stimulation (tnVNS) group and a taVNS group, with 12 rats in each group. The suture-occluded method was adopted to prepare MCAO rat model. The auricular rim was stimulated in the tnVNS group and the concha stimulated in the taVNS group, 2 mA in intensity, 10 Hz in frequency, 30 min each time, once a day, for 14 days consecutively. The nerve functional assessment was recorded in each group. The expressions of nicotinic acetylcholine receptor (α7nAchR) in the cerebral ischemic penumbra and the spleen were detected by using Western blot. With the immunofluorescence, the expressions of GFAP and MAP2 were detected. After modeling, compared with the sham-operation group, the nerve functional score was increased in the model group, the tnVNS group and the taVNS group (P<0.01), suggesting the success of modeling. After treatment, the score was increased in the model group (P<0.01) as compared with the sham-operation group. Compared with the model group, the neurological deficit score was reduced in the taVNS group (P<0.01). Compared with the sham-operation group, GFAP expression was increased and MAP2 expression was reduced remarkably in the cerebral ischemic penumbra in the model group (P<0.05). In comparison with the model group, GFAP expression was reduced, while MAP2 expression was increased remarkably in the cerebral ischemic penumbra in the taVNS group (P<0.05). There were no significant differences in the abovementioned indexes between the model group and tnVNS group (P>0.05). The differences in the expression of α7nAchR in the cerebral ischemic penumbra and the spleen had no statistical significance among groups (P>0.05). TaVNS is effective on neuroprotection in MCAO rats, which may be related to its function of inhibition of GFAP expression and promotion of MAP2 expression in the ischemic penumbra.

Investigation of brain damage mechanism in middle cerebral artery occlusion/reperfusion rats based on i-TRAQ quantitative proteomics.

The objective of this study is to analyze the differential protein expression profile in cerebral cortex of rats with middle cerebral ischemia/reperfusion (MCAO/R), explore the brain damage mechanism of MCAO/R at protein level, and provide experimental foundation for searching specific marker proteins of MCAO/R. Rat model of MCAO/R was established by modified suture-occluded method, and the model was evaluated by the results of brain 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin-eosin (HE) staining. Cerebral cortex of rats from sham-operated group (Sham) and MCAO/R groups was used for FASP enzymatic hydrolysis, i-TRAQ quantitative labeling, and reverse-phase liquid chromatography purification and separation. Orbitrap Q Exactive mass spectrometry was used for qualitative and quantitative analyses of total differential protein expression profiles. MCAO/R rats had obvious cerebral infarction lesions, and the relative surface area of cerebral infarction was significantly different compared with sham rats, suggesting that MCAO/R rat model was successfully prepared. There were 199 significant difference proteins (MCAO/R vs Sham, p < 0.05, |fold change|> 1.2), including 104 up-regulated proteins and 95 down-regulated proteins. Gene ontology (GO) enrichment analysis showed that the up-regulated proteins were mainly concentrated in the biological processes of positive regulation of NF-κB transcription and I-κB kinase-NF-κB, etc. Down-regulated proteins were mainly concentrated in long-term synaptic potentiation, cellular response to DNA damage stimulus, etc. KEGG pathway analysis showed that the pathway involved in differential proteins includes oxidative phosphorylation, metabolic pathway, and Ras signaling pathway. Network analysis of differential proteins showed that Alb, ndufb5, ndufs7, ApoB, Cdc42, Ndufa3, Igf1r, P4hb, Mbp, Gc, Nme1, Akt2, and other proteins may play an important role in regulating oxidative stress, apoptosis, and inflammatory response in MCAO/R. Quantitative proteomics based on i-TRAQ labeling reveals the effect of inflammation and apoptosis in brain damage mechanism of MCAO/R. Besides, this research provide some experimental foundation for search and determination of potential therapeutic targets of MCAO/R.

Taohong Siwu Decoction Ameliorates Ischemic Stroke Injury Via Suppressing Pyroptosis.

Objective: Taohong Siwu decoction (THSWD) is one of the classic prescriptions for promoting blood circulation and removing blood stasis, and it has a good therapeutic effect on ischemic stroke. We sought to explore the therapeutic effects of THSWD on pyroptosis in rats with middle cerebral artery occlusion-reperfusion (MCAO/R). Methods: MCAO/R model of rats were established by suture-occluded method. MCAO/R rats were randomly divided into five groups, which were model group, nimodipine group, THSWD high, medium and low dose group (18, 9, and 4.5 g/kg, respectively), rats of sham group without thread embolus. All rats were treated by intragastric administration for 7 days. We detected the level of inflammatory factors. NLRP3 and Caspase-1 were detected by immunofluorescence. Western blot was used to detect NLRP3, Caspase-1, ASC, and GSDMD in penumbra. Also, the expression of TXNIP, HMGB1, toll-like receptors (TLR4), NF-κB, and MAPK were detected. Results: THSWD treatment improved the behavioral function and brain pathological damage. These results showed that the levels of TNF-α, TGF-β, IL-2, IL-6, IL-1β, and IL-18 were significantly reduced in THSWD treatment groups. THSWD could significantly decrease the expression levels of NLRP3, Caspase-1, Caspase-1 p10, ASC, TXNIP, GSDMD, HMGB1, TLR4/NFκB, p38 MAPK, and JNK in penumbra. Conclusion: Our results showed that THSWD could reduce the activation level of NLRP3 inflammatory corpuscle, down-regulate GSDMD, and inhibit pyroptosis in MCAO/R rats. These may be affected by inhibiting HMGB1/TLR4/NFκB, MAPK signaling pathways.

Neuroprotection by cattle encephalon glycoside and ignotin beyond the time window of thrombolysis in ischemic stroke.

Cattle encephalon glycoside and ignotin (CEGI) injection is known as a multi-target neuroprotective drug that contains numerous liposoluble molecules, such as polypeptides, monosialotetrahexosyl ganglioside (GM-1), free amino acids, hypoxanthine and carnosine. CEGI has been approved by the Chinese State Food and Drug Administration and widely used in the treatments of various diseases, such as stroke and Alzheimer's disease. However, the neuroprotective effects of CEGI beyond the time window of thrombolysis (within 4.5 hours) on acute ischemic stroke remain unclear. This study constructed a rat middle cerebral artery occlusion model by suture-occluded method to simulate ischemic stroke. The first daily dose was intraperitoneally injected at 8 hours post-surgery and the CEGI treatments continued for 14 days. Results of the modified five-point Bederson scale, beam balance test and rotameric test showed the neurological function of ischemic stroke rats treated with 4 mL/kg/d CEGI improved significantly, but the mortality within 14 days did not change significantly. Brain MRI and 2,3,5-triphenyltetrazolium chloride staining confirmed that the infarct size in the 4 mL/kg/d CEGI-treated rats was significantly reduced compared with ischemic insult only. The results of transmission electron microscopy and double immunofluorescence staining showed that the hippocampal neuronal necrosis in the ischemic penumbra decreased whereas the immunopositivity of new neuronal-specific protein doublecortin and the percentage of Ki67/doublecortin positive cells increased in CEGI-treated rats compared with untreated rats. Our results suggest that CEGI has an effective neuroprotective effect on ischemic stroke when administered after the time window of thrombolysis. The study was approved by the Animal Ethics Committee of The Third Military Medical University, China.

MicroRNA-130a regulates neurological deficit and angiogenesis in rats with ischaemic stroke by targeting XIAP.

MicroRNAs (miRNAs) have already been proposed to be implicated in the development of ischaemic stroke. We aim to investigate the role of miR‐130a in the neurological deficit and angiogenesis in rats with ischaemic stroke by regulating X‐linked inhibitor of apoptosis protein (XIAP). Middle cerebral artery occlusion (MCAO) models were established by suture‐occluded method, and MCAO rats were then treated with miR‐130a mimics/inhibitors or/and altered XIAP for detection of changes of rats’ neurological function, nerve damage and angiogenesis in MCAO rats. The oxygen‐glucose deprivation (OGD) cellular models were established and respectively treated to determine the roles of miR‐130a and XIAP in neuronal viability and apoptosis. The expression levels of miR‐130a and XIAP in brain tissues of MCAO rats and OGD‐treated neurons were detected. The binding site between miR‐130a and XIAP was verified by luciferase activity assay. MiR‐130a was overexpressed while XIAP was down‐regulated in MCAO rats and OGD‐treated neurons. In animal models, suppressed miR‐130a improved neurological function, alleviated nerve damage and increased new vessels in brain tissues of rats with MCAO. In cellular models, miR‐130a inhibition promoted neuronal viability and suppressed apoptosis. Inhibited XIAP reversed the effect of inhibited miR‐130a in both MCAO rats and OGD‐treated neurons. XIAP was identified as a target of miR‐130a. Our study reveals that miR‐130a regulates neurological deficit and angiogenesis in rats with MCAO by targeting XIAP.

Lidocaine improves cerebral ischemia-reperfusion injury in rats through cAMP/PKA signaling pathway

Influence of lidocaine on rats with cerebral ischemia-reperfusion injury (CIRI) was studied to explore its mechanism of action. A total of 30 Sprague-Dawley rats were randomly divided into control group and model group, and the rat model of CIRI was prepared by the suture-occluded method in the model group. Then the rats in the model group were randomly assigned into the model group (n=10) and the lidocaine group (n=10). The neurological function score of rats was evaluated, and the levels of serum B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in rats were determined using ELISA. TUNEL assay was performed to detect the neuronal apoptosis in the brain of rats. The messenger ribonucleic acid (mRNA) and protein expression levels of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) were measured via RT-PCR and western blotting, respectively. Compared with those in the control group, the rats in the model group had an elevated neurological function score, a raised level of Bcl-2, but a reduced level of Bax in the serum, an obviously increased rate of neuronal apoptosis in the brain and decreased mRNA and protein levels of cAMP and PKA in cerebral tissues. The rats in lidocaine group had a lower neurological function score, a lower level of Bcl-2, but a higher level of Bax in the serum, an evidently lower rate of neuronal apoptosis in the brain and higher mRNA and protein levels of cAMP and PKA in cerebral tissues than those in the model group. Lidocaine can improve the neurological function of rats with CIRI and inhibit neuronal apoptosis in the brain, and its mechanism of action may be related to the activation of the cAMP/PKA signaling pathway.

Effect of An-pressing manipulation on post-stroke muscle spasticity in rats and its mechanism study

To explore the mechanism of An-pressing manipulation in improving post-stroke muscle spasticity, by observing the changes of γ-aminobutyric acid (GABA) and glycine (Gly) in plasma and gray matter of L1-L3 spinal cord anterior horn in post-stroke rats with muscle spasticity after An-pressing manipulation intervention. Ten of 80 adult male Sprague-Dawley (SD) rats were randomly selected as the blank group, and the remaining 70 were used for modeling. The middle cerebral artery occlusion (MCAO) rat model was established by insertion suture occlusion method in the left external carotid artery. Thirty rats with a Longa neurological score of 2-3 points and a modified Ashworth spasticity scale score of 1−, 1+, or 2 were included in the experiment. Using the random number table method, the 30 successfully modeled rats were randomly divided into a model group, an An-pressing tendon group and an An-pressing muscle belly group. Two days after modeling, rats in the An-pressing tendon group and An-pressing muscle belly group received An-pressing manipulation on the tendon and belly of quadriceps femoris muscle respectively, with the pressure of (350±50) g and the frequency of 5 s/time, 15 min per session, once a day for 5 continuous days. After the 5th treatment, the tension of the rat quadriceps femoris muscle was evaluated using the modified Ashworth spasticity scale. The Gly levels in rat plasma and L1-L3 segments of spinal cord were determined by enzyme-linked immunosorbent assay (ELISA). The GABA levels in rat plasma and L1-L3 segments of spinal cord were measured by high performance liquid chromatography (HPLC). The decrease in rat muscle tension scored by the modified Ashworth spasticity scale in the An-pressing tendon group was more significant than that in the An-pressing muscle belly group (P<0.01); the increases in Gly and GABA levels in the rat plasma and L1-L3 segments of spinal cord were more significant in the An-pressing tendon group than those in the An-pressing muscle belly group (all P<0.01). Based on the theory of ‘anti-stretch reflex’ of tendon organs, the use of An-pressing manipulation to induce the ‘anti-stretch reflex’ by stimulating the tendon organs can improve the muscle spasticity of rats, which is better than An-pressing the muscle belly. Increased levels of Gly and GABA in rat plasma and L1-L3 segments of spinal cord may be one mechanism of An-pressing manipulation to improve muscle spasticity by stimulating tendon organs.

Effect of ginsenosides on serous metabonomic profiles in cerebral ischemia-reperfusion rats based on ~1H-NMR

Serum metabonomic profiles of the model of focal cerebral ischemia reperfusion is established with the suture-occluded method by Longa to study the effect of ginsenosides. In this study, 48 rats were randomly divided into six groups: sham-operated group, pathological model group, positive drug group(6 mg·kg~(-1)·d~(-1)) and high, medium, low-dose ginsenosides groups(200, 100, 50 mg·kg~(-1)·d~(-1)). They are given intragastric administration respectively with same amount of 0.5% CMC-Na,nimodipine and ginsenoside for 5 days. At 2 h after the final administration, the model was established with the suture-occluded method, and free radical-scavenging activity changes of ginsenoside were observed by maillard reaction, and Longa was possible used as a renoprotective agent-occluded method. At the end of 24 h after the reperfusion, the hemolymph of rats in each group was collected, and the ~1H-NMR spectrum was collected after being treated by certain methods, and analyzed by principal component analysis(PCA). Compared with sham-operated group, pathological model group showed significant increases in the levels of lactate, glutamate, taurine, choline, glucose and methionine, but decreases in the levels of 3-hydroxybutyrate and phosphocreatine/creatine in serum. After treatment with ginsenosides, lipid, 3-hydroxybutyrate and phosphocreatine/creatine were increased in the serum of ginsenosides group rats, but with decreases in lactate and glutamate. The results showed that ginsenosides could regulate metabolic disorders in rats with focal cerebral ischemia reperfusion, and promote a recovery in the process of metabolism. It's helpful to promote the metabolic changes in rats with focal cerebral ischemia reperfusion via ~1H-NMR, and lay a foundation to develop ginsenosides as a new drug to treat ischemic cerebral paralysis.

Effects of cluster needling of scalp acupuncture on neurofilament protein 200 and signal transducer and activator of transcription 3 in rats with acute cerebral ischemia

Abstract Objective Cluster needling at scalp acupoints has showed satisfying effects with acute cerebral ischemia in clinic whereas the mechanisms are not yet clear completely. This study investigated the influence of cluster needling at scalp acupoints on neurological function, as well as on neurofilament protein 200 (NF200) and signal transducer and activator of transcription 3 (STAT3) expression, in rats with acute cerebral ischemia. Methods Fifty-four Sprague-Dawley rats were randomly assigned in equal numbers to the false operation (group F), model (group M), or cluster needling scalp acupuncture (group C) groups. Each group was divided into three subgroups, of six rats each, by acupuncture treatment time (24 h, 7 days, and 14 days). The rat local cerebral ischemia model was prepared using a modified suture occlusion method. Group C rats were treated by cluster needling scalp acupuncture, while groups F and M did not receive acupuncture treatment. Neurological effects were evaluated using the Longa score. NF200 and STAT3 expression were measured by western blotting. Results At 24 h, there were no statistical difference between group C and group M in nerve function (P > .05). On days 7 and 14, nerve function scores in group C were significantly lower than that in group M (respectively were P .05). Conclusion Cluster needling scalp acupuncture were efficient in improving nerve function scores in rats with cerebral ischemia, and promoting the recovery of motor function. These improvements were associated with increases in NF200 and STAT3 expression.

Triptolide mediates Wnt/β-catenin signalling pathway to reduce cerebral ischemia-reperfusion injury in rats.

Triptolide, extracted from Chinese medicinal materials Tripterygium wilfordii Hook F (TwHF), has immunosuppressive, anti-inflammatory and anti-tumour effects. The purpose of this study was to examine whether triptolide has the neuroprotective effect on cerebral ischemia-reperfusion (I/R) injury and to explore its possible mechanism. The rat model of focal cerebral I/R was established by the suture-occluded method. The SD rats were randomly divided into five groups: sham operation group (Sham group), ischemia-reperfusion model group (I/R group), low concentration of triptolide group (12.5 mg/kg, TL-L group), medium concentration of triptolide group (25 mg/kg, TL-M group) and high concentration of triptolide group (50 mg/kg, TL-H group). The neurological function of the rats was scored, the degree of brain oedema was detected by the dry-wet method, and the cerebral infarction area was determined by TTC staining. Nissl staining was used to detect neuronal damage. The contents of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) were also detected. Meanwhile, the expression level of proteins related to Wnt/-catenin signalling pathway was measured by Western blot. Compared with the I/R group, cerebral oedema, infarct volume, neurological impairment, the contents of MDA and ROS were reduced, while the SOD level was increased in the TL-L, TL-M, and TL-H groups. The results of Nissl staining showed that triptolide could reduce the nerve cell injury caused by cerebral I/R. In addition, the results of Western blot confirmed that the expression of Wnt1, -catenin, c-Myc, and Cyclin-D1 were down-regulated after triptolide intervention, that is, inhibited the activation of Wnt/-catenin signalling pathway. Triptolide mediates Wnt/-catenin signalling pathway to alleviate cerebral I/R injury in rats. This study provides ideas and experimental basis for the treatment of ischemic stroke patients.