Role of curcumin-mediated inhibition of inflammation in alleviating brain ischemia-reperfusion synaptic injury / 公共卫生与预防医学

Objective To investigate the potential effect and mechanism of curcumin in inhibiting synaptic injury in the cortex of rats with cerebral ischemia-reperfusion. Methods Sprague-Dawley rats were divided into sham-operated group, model group, low-dose curcumin (50 mg/kg) group, and high-dose curcumin (100 mg/kg) group. A model of middle cerebral artery occlusion for 2 hours and reperfusion for 24 hours was constructed, and curcumin was administered. Based on the neurological function score, the effects of curcumin on cerebral infarct volume, synaptic ultrastructure changes, inflammatory cell infiltration, and the expression of NLRP3, Caspase-1, Synapsin1, and CAMKⅡ were observed after the end of the animal treatment. Results The neurological function scores were 0, 3.25±0.43, 2.50±0.50, and 1.50±0.50 for the sham-operated group, model group, low-dose curcumin group, and high-dose curcumin group, respectively. The percentage of cerebral infarct volume was 0, (38.89±2.21)%, (33.48±1.77)%, and (23.69±2.19)%, respectively. Compared with the sham operation group, the model group had severe synaptic ultrastructure damage, extensive inflammatory cell infiltration, significantly increased expression of Caspase-1 and NLRP3 (P < 0.5), and significantly decreased expression of Synapsin1 and CAMKⅡ (P < 0.5). Curcumin treatment significantly inhibited synaptic damage, reduced inflammatory cell infiltration, decreased the expression of Caspase-1 and NLRP3 (P < 0.5), and increased the expression of Synapsin1 and CAMKII (P < 0.5), when compared with the model group. Conclusion Ischemia-reperfusion-mediated synaptic injury in rat brain triggers an inflammatory response in cortical nerve cells, and curcumin alleviates synaptic damage and reduces brain injury by inhibiting inflammatory factor levels.

The antioxidant effect of preischemic dexmedetomidine in a rat model: increased expression of Nrf2/HO-1 via the PKC pathway

Abstract Background The precise underlying mechanism of antioxidant effects of dexmedetomidine-induced neuroprotection against cerebral ischemia has not yet been fully elucidated. Activation of Nuclear factor erythroid 2-related factor (Nrf2) and Heme Oxygenase-1 (HO-1) represents a major antioxidant-defense mechanism. Therefore, we determined whether dexmedetomidine increases Nrf2/HO-1 expression after global transient cerebral ischemia and assessed the involvement of Protein Kinase C (PKC) in the dexmedetomidine-related antioxidant mechanism. Methods Thirty-eight rats were randomly assigned to five groups: sham (n = 6), ischemic (n = 8), chelerythrine (a PKC inhibitor; 5 mg.kg-1 IV administered 30 min before cerebral ischemia) (n = 8), dexmedetomidine (100 µg.kg-1 IP administered 30 min before cerebral ischemia (n = 8), and dexmedetomidine + chelerythrine (n = 8). Global transient cerebral ischemia (10 min) was applied in all groups, except the sham group; histopathologic changes and levels of nuclear Nrf2 and cytoplasmic HO-1 were examined 24 hours after ischemia insult. Results We found fewer necrotic and apoptotic cells in the dexmedetomidine group relative to the ischemic group (p< 0.01) and significantly higher Nrf2 and HO-1 levels in the dexmedetomidine group than in the ischemic group (p< 0.01). Additionally, chelerythrine co-administration with dexmedetomidine attenuated the dexmedetomidine-induced increases in Nrf2 and HO-1 levels (p< 0.05 and p< 0.01, respectively) and diminished its beneficial neuroprotective effects. Conclusion Preischemic dexmedetomidine administration elicited neuroprotection against global transient cerebral ischemia in rats by increasing Nrf2/HO-1 expression partly via PKC signaling, suggesting that this is the antioxidant mechanism underlying dexmedetomidine-mediated neuroprotection.

Celastrol targeting Nedd4 reduces Nrf2-mediated oxidative stress in astrocytes after ischemic stroke / 药物分析学报

Stroke is the second leading cause of death worldwide,and oxidative stress plays a crucial role.Celastrol exhibits strong antioxidant properties in several diseases;however,whether it can affect oxidation in cerebral ischemic-reperfusion injury(CIRI)remains unclear.This study aimed to determine whether celastrol could reduce oxidative damage during CIRI and to elucidate the underlying mechanisms.Here,we found that celastrol attenuated oxidative injury in CIRI by upregulating nuclear factor E2-related factor 2(Nrf2).Using alkynyl-tagged celastrol and liquid chromatography-tandem mass spectrometry,we showed that celastrol directly bound to neuronally expressed developmentally downregulated 4(Nedd4)and then released Nrf2 from Nedd4 in astrocytes.Nedd4 promoted the degradation of Nrf2 through K48-linked ubiquitination and thus contributed to astrocytic reactive oxygen species production in CIRI,which was significantly blocked by celastrol.Furthermore,by inhibiting oxidative stress and astrocyte activation,celastrol effectively rescued neurons from axon damage and apoptosis.Our study uncovered Nedd4 as a direct target of celastrol,and that celastrol exerts an antioxidative effect on as-trocytes by inhibiting the interaction between Nedd4 and Nrf2 and reducing Nrf2 degradation in CIRI.

An improved 4-vessel intermittent occlusion method for establishing rat models of global cerebral ischemia-reperfusion injury / 南方医科大学学报

OBJECTIVE@#To improve the classical 4-vessel occlusion (4VO) model established by Pulsinelli and Brierley.@*METHODS@#Thirty-two male SD rats were randomized into sham operation group, I4VO-Con10 group, I4VO-Int10 group and I4VO-Int15 group. The sham surgery group underwent exposure of the bilateral vertebral arteries and carotid arteries without occlusion to block blood flow. The I4VO-Con10 group experienced continuous ischemia by occluding the bilateral vertebral arteries and carotid arteries for 10 minutes followed by reperfusion for 24 hours. The I4VO-Int10 and I4VO-Int15 groups were subjected to intermittent ischemia. The I4VO- Int10 group underwent 5 minutes of ischemia, followed by 5 minutes of reperfusion and another 5 minutes of ischemia, and then reperfusion for 24 hours. The I4VO-Int15 group experienced 5 minutes of ischemia followed by two cycles of 5 minutes of reperfusion and 5 minutes of ischemia, and then reperfusion for 24 hours. The regional cerebral blood flow (rCBF) was monitored with laser Doppler scanning, and survival of the rats was observed. HE staining was used to observe hippocampal pathologies to determine the optimal method for modeling. Another 48 rats were randomized into 6 groups, including a sham operation group and 5 model groups established using the optimal method. The 5 I4VO model groups were further divided based on the reperfusion time points (1, 3, 7, 14, and 28 days) into I4VO-D1, I4VO-D3, I4VO-D7, I4VO- D14, and I4VO- D28 groups. Body weight changes and survival of the rats were recorded. HE staining was used to observe morphological changes in the hippocampal, retinal and optic tract tissues. The Y-maze test and light/dark box test were used to evaluate cognitive and visual functions of the rats in I4VO-D28 group.@*RESULTS@#Occlusion for 5 min for 3 times at the interval of 5 min was the optimal method for 4VO modeling. In the latter 48 rats, the body weight was significantly lower than that of the sham-operated rats at 1, 3, 7, 14 and 28 days after modeling without significant difference in survival rate among the groups. The rats with intermittent vessel occlusion exhibited progressive deterioration of hippocampal neuronal injury and neuronal loss. Cognitive impairment was observed in the rats in I4VO-D28 group, but no obvious ischemic injury of the retina or the optic tract was detected.@*CONCLUSION@#The improved 4VO model can successfully mimic the main pathological processes of global cerebral ischemia-reperfusion injury without causing visual impairment in rats.

Protective Effect of Huazhuo Jiedu Huoxue Tongluo Prescription on Cerebral Ischemia-reperfusion Injury via Regulating Autophagy Based on JNK Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Huazhuo Jiedu Huoxue Tongluo prescription in alleviating cerebral ischemia-reperfusion injury via regulating nerve cell autophagy based on c-Jun N-terminal kinase（JNK）signaling pathway . MethodSixty SD rats were randomly divided into 6 groups： sham group， middle cerebral artery occlusion/reperfusion （MCAO/R） group （model group）， Huazhuo Jiedu Huoxue Tongluo prescription group ［traditional Chinese medicine （TCM） group（25.0 g·kg-1）］， JNK inhibitor SP600125 （SP） group（5 mg·kg-1）， TCM+SP group and JNK agonist Anisomycin （Ani） group（15 mg·kg-1）. After 24 h of modeling， TCM group and TCM+SP group were given TCM decoction （ig） for 3 consecutive days， and the other groups were given equal volume of normal saline （ig）. Neurological deficit was evaluated by neurological function score and cerebral infarct volume was determined by 2，3，5-triphenyltetrazole chloride （TTC） staining. Hematoxylin-eosin （HE） staining and Nissl staining were used to observe the structural changes of brain tissue and the damage of neurons， respectively. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay （TUNEL） was performed to detect cell apoptosis. The ultrastructure of autophagosomes was observed by transmission electron microscope. Western blot was employed to detect the protein expressions of microtubule-associated protein 1 light chain 3A/B （LC3A/B）， autophagy related 5 （Atg5）， the ortholog of yeast Atg6 （Beclin1）， p62， B-cell lymphoma 2 （Bcl-2）， JNK， phosphorylated （p）-JNK and c-Jun in brain tissue. The mRNA expressions of LC3A/B， Beclin1， p62， Atg5， Bcl-2， JNK and c-Jun were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the sham group， the model group had elevated neurological deficit score （P<0.05）， enlarged cerebral infarct volume （P<0.05）and typical infarction manifestations formed in hippocampal region and its surrounding brain tissue. In addition， there were a large number of neuronal cell degeneration， necrosis， liquefaction， nucleus pyknosis and deep staining， and inflammatory cell infiltration in the cortex in the model group， and severe swelling of mitochondria， lysosomes， autophagosomes and autophagolysosomes were clearly seen under electron microscope. TUNEL positive cells were increased （P<0.05）， and cell apoptosis was severe. The nuclear membrane and nucleolus of neurons in brain tissue were blurred with discontinuous processes， and Nissl bodies in cytoplasm were stained light with reduced number. Western blot revealed that the model group had up-regulated protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun in brain tissue （P<0.05）， while down-regulated protein expressions of p62 and Bcl-2 （P<0.05）as compared with the sham group. Real-time PCR indicated that the mRNA expressions of LC3A/B， Beclin1， Atg5， JNK and c-Jun in the model group were higher （P<0.05） while the mRNA expressions of p62 and Bcl-2 were lower （P<0.05） than those in the sham group. Compared with the conditions in model group， the neurological deficit scores of TCM， SP and TCM+SP groups were lowered （P<0.05）， and the cerebral infarct volume was reduced （P<0.05）， with improved pathological status of brain tissue， especially in the TCM group. Furthermore， there were abundant and basically normal mitochondrial cristae， slightly dilated endoplasmic reticulum， slightly swollen golgi apparatus， slightly fused nuclear membrane， and few visible lysosomes， autophagosomes and autophagolysosomes. TUNEL positive cells were decreased （P<0.05）， displaying reduced apoptosis， especially in the TCM group. The nucleolus and nuclear membrane of neurons in brain tissue were discernible， and Nissl bodies in cytoplasm was reduced to a certain degree as compared with those in the model group. Western blot showed a decrease in the protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun in the TCM group ，the SP group，and the TCM+SP group（P<0.05），while an increase in the protein expressions of p62 in the TCM group and SP group（P<0.05），and an increase in the protein expressions of Bcl-2 in the TCM group and TCM+SP group. By Real-time PCR， the mRNA expressions of LC3A， LC3B， Beclin1， Atg5， JNK and c-Jun had a down-regulation（P<0.05） while the mRNA expression of p62 a up-regulation in the TCM group ，the SP group，and the TCM+SP group（P<0.05），and the mRNA expression of Bcl-2 a up-regulation in the TCM group and the TCM+SP group（P<0.05）.Scores of the Ani group were raised （P<0.05）， and infarct volume was increased significantly， with aggravated neuronal cell necrosis and obvious inflammatory infiltration. Moreover， there were neuronal nuclear membrane fusion with abnormal protrusion， increased heterochromatin aggregation in edge， severe mitochondrial swelling， endoplasmic reticulum expansion， increased lysosomes， increased intracytoplasmic vesicles， and visible autophagosomes and autophagolysosomes. TUNEL positive cells were increased （P<0.05）， representing severe apoptosis. The number of Nissl bodies dropped with light staining， and the nucleolus and nuclear membrane were blurred. Real-time PCR found that the mNRA expressions of Atg5， c-Jun， JNK were up-regulated （P<0.05），while Beclin1， p62， Bcl-2 were were down-regulated in the Ani group （P<0.05）. Compared with the TCM group and SP group，the protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK， c-Jun were decreased，and p62， Bcl-2 were increased in the Ani group（P<0.05）. Compared with the TCM group，the mRNA expressions of JNK mRNA had a down-regulation in the SP group and TCM+SP group，while LC3A， LC3B， Atg5， c-Jun， JNK had an up-regulation（P<0.05） and Bcl-2 had a down-regulation in the Ani group（P<0.05）. Compared with the SP group，the mRNA expressions of Atg5， c-Jun， JNK had an up-regulation（P<0.05）， and Beclin1， p62， Bcl-2 had a down-regulation in the Ani group（P<0.05）. ConclusionHuazhuo Jiedu Huoxue Tongluo prescription significantly up-regulates the protein and mRNA expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun， and down-regulates the protein and mRNA expressions of p62 and Bcl-2， suggesting that the prescription can inhibit autophagy through JNK signaling pathway to reduce ischemia/reperfusion injury in rats.

Mechanism of Chinese Medicine Saponins in Treatment of Cerebral Ischemia-reperfusion Based on Literature Analysis / 中国实验方剂学杂志

Cerebral ischemia-reperfusion is a serious cerebrovascular disease with high morbidity and mortality. In recent years， reperfusion therapy based on thrombolysis and thrombectomy has been the main treatment method for patients with ischemic stroke. Numerous studies have shown that Chinese medicine saponins can effectively interfere with cerebral ischemia-reperfusion in a multi-target and multi-way manner， which have great potential on the treatment and prevention of cerebral ischemia-reperfusion. Taking China National Knowledge Infrastructure （CNKI） literature database and Wanfang literature database as the analysis sources， this paper used SPSS statistics to summarize the number of papers on the treatment of cerebral ischemia-reperfusion with Chinese medicine saponins and CiteSpace to conduct cluster analysis on the high-frequency keywords of the research， thereby expounding the research hotspots and research status of Chinese medicine saponins in the treatment of cerebral ischemia-reperfusion. Based on literature analysis and summary of animal experiments on the treatment of cerebral ischemia-reperfusion with Chinese medicine saponins in the past two decades， Chinese medicine saponins exerted effects by anti-inflammation， inhibition of oxidative stress， immune regulation， protection of nerve cells， inhibition of thrombosis， promotion of thrombolysis， protection of mitochondria， blood-brain barrier repairing， and other ways. The specific mechanism， therapeutic effect， and signaling pathway of each Chinese medicine saponin have been summarized in this study， which provide a theoretical basis for the in-depth research， new drug development， and clinical application of Chinese medicine saponins for the treatment of cerebral ischemia- reperfusion.

Effect of Xiaoxuming Decoction on synaptic plasticity following acute cerebral ischemia-reperfusion in rats / 中国中药杂志

This study aims to explore the effect of Xiaoxuming Decoction on synaptic plasticity in rats with acute cerebral ischemia-reperfusion. A rat model of cerebral ischemia-reperfusion injury was established by middle cerebral artery occlusion(MCAO). Rats were randomly assigned into a sham group, a MCAO group, and a Xiaoxuming Decoction(60 g·kg~(-1)·d~(-1)) group. The Longa score was rated to assess the neurological function of rats with cerebral ischemia for 1.5 h and reperfusion for 24 h. The 2,3,5-triphenyltetrazolium chloride(TTC) staining and hematoxylin-eosin(HE) staining were employed to observe the cerebral infarction and the pathological changes of brain tissue after cerebral ischemia, respectively. Transmission electron microscopy was employed to detect the structural changes of neurons and synapses in the ischemic penumbra, and immunofluorescence, Western blot to determine the expression of synaptophysin(SYN), neuronal nuclei(NEUN), and postsynaptic density 95(PSD95) in the ischemic penumbra. The experimental results showed that the modeling increased the Longa score and led to cerebral infarction after 24 h of ischemia-reperfusion. Compared with the model group, Xiaoxuming Decoction intervention significantly decreased the Longa score and reduced the formation of cerebral infarction area. The modeling led to the shrinking and vacuolar changes of nuclei in the brain tissue, disordered cell arrangement, and severe cortical ischemia-reperfusion injury, while the pathological damage in the Xiaoxuming Decoction group was mild. The modeling blurred the synaptic boundaries and broadened the synaptic gap, while such changes were recovered in the Xiaoxuming Decoction group. The modeling decreased the fluorescence intensity of NEUN and SYN, while the intensity in Xiaoxuming Decoction group was significantly higher than that in the model group. The expression of SYN and PSD95 in the ischemic penumbra was down-regulated in the model group, while such down-regulation can be alleviated by Xiaoxuming Decoction. In summary, Xiaoxuming Decoction may improve the synaptic plasticity of ischemic penumbra during acute cerebral ischemia-reperfusion by up-regulating the expression of SYN and PSD95.

Quality control mechanism of mitochondria by 3,4-dihydroxybenzaldehyde through OGT-PINK1 pathway / 中国中药杂志

Based on the O-GlcNAc transferase(OGT)-PTEN-induced putative kinase 1(PINK1) pathway, the mechanism of 3,4-dihydroxybenzaldehyde(DBD) on mitochondrial quality control was investigated. Middle cerebral artery occlusion/reperfusion(MCAO/R) rats were established. SD rats were randomized into sham operation group(sham), model group(MCAO/R), DBD-L group(5 mg·kg~(-1)), and DBD-H group(10 mg·kg~(-1)). After 7 days of administration(ig), MCAO/R was induced in rats except the sham group with the suture method. Twenty-four h after reperfusion, the neurological function and the percentage of cerebral infarct area were measured. Based on hematoxylin and eosin(HE) staining and Nissl staining, the pathological damage of cerebral neurons was examined. Then the ultrastructure of mitochondria was observed under the electron microscope, and the co-localization of light chain-3(LC3), sequestosome-1(SQSTM1/P62), and Beclin1 was further detected by immunofluorescence staining. It has been reported that the quality of mitochondria can be ensured by inducing mitochondrial autophagy through the OGT-PINK1 pathway. Therefore, Western blot was employed to detect the expression of OGT, mitophagy-related proteins PINK1 and E3 ubiquitin ligase(Parkin), and mitochondrial kinetic proteins dynamin-like protein 1(Drp1) and optic atrophy 1(Opa1). The results showed that MCAO/R group had neurological dysfunction, large cerebral infarct area(P<0.01), damaged morphological structure of neurons, decreased number of Nissl bodies, mitochondrial swelling, disappearance of mitochondrial cristae, decrease of cells with LC3 and Beclin1, rise of cells with P62(P<0.01), inhibited expression of OGT, PINK1, and Parkin, up-regulated expression of Drp1, and down-regulated expression of Opa1 compared with the sham group(P<0.01). However, DBD improved the behavioral deficits and mitochondrial health of MCAO/R rats, as manifested by the improved morphology and structure of neurons and mitochondria and the increased Nissl bodies. Moreover, DBD increased cells with LC3 and Beclin1 and decreased cells with P62(P<0.01). In addition, DBD promoted the expression of OGT, PINK1, Parkin, and Opa1 and inhibited the expression of Drp1, enhancing mitophagy(P<0.05, P<0.01). In conclusion, DBD can trigger PINK1/Parkin-mediated brain mitophagy through the OGT-PINK1 pathway, which plays a positive role in maintaining the health of the mitochondrial network. This may be a mitochondrial therapeutic mechanism to promote nerve cell survival and improve cerebral ischemia/reperfusion injury.

Chrysin alleviates cerebral ischemia-reperfusion injury by inhibiting ferroptosis in rats / 中国中药杂志

The purpose of this study is to investigate whether chrysin reduces cerebral ischemia-reperfusion injury(CIRI) by inhi-biting ferroptosis in rats. Male SD rats were randomly divided into a sham group, a model group, high-, medium-, and low-dose chrysin groups(200, 100, and 50 mg·kg~(-1)), and a positive drug group(Ginaton, 21.6 mg·kg~(-1)). The CIRI model was induced in rats by transient middle cerebral artery occlusion(tMCAO). The indexes were evaluated and the samples were taken 24 h after the operation. The neurological deficit score was used to detect neurological function. The 2,3,5-triphenyl tetrazolium chloride(TTC) staining was used to detect the cerebral infarction area. Hematoxylin-eosin(HE) staining and Nissl staining were used to observe the morphological structure of brain tissues. Prussian blue staining was used to observe the iron accumulation in the brain. Total iron, lipid pero-xide, and malondialdehyde in serum and brain tissues were detected by biochemical reagents. Real-time quantitative polymerase chain reaction(RT-qPCR), immunohistochemistry, and Western blot were used to detect mRNA and protein expression of solute carrier fa-mily 7 member 11(SLC7A11), transferrin receptor 1(TFR1), glutathione peroxidase 4(GPX4), acyl-CoA synthetase long chain family member 4(ACSL4), and prostaglandin-endoperoxide synthase 2(PTGS2) in brain tissues. Compared with the model group, the groups with drug intervention showed restored neurological function, decreased cerebral infarction rate, and alleviated pathological changes. The low-dose chrysin group was selected as the optimal dosing group. Compared with the model group, the chrysin groups showed reduced content of total iron, lipid peroxide, and malondialdehyde in brain tissues and serum, increased mRNA and protein expression levels of SLC7A11 and GPX4, and decreased mRNA and protein expression levels of TFR1, PTGS2, and ACSL4. Chrysin may regulate iron metabolism via regulating the related targets of ferroptosis and inhibit neuronal ferroptosis induced by CIRI.

Protective effects of three kinds of borneol on different brain regions in acute cerebral ischemia/reperfusion model rats / 中国中药杂志

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.

Effective constituents of essential oil from Gleditsiae Fructus Abnormalis and anti-cerebral ischemia/reperfusion injury mechanism: based on GC-MS, network pharmacology, and experimental verification / 中国中药杂志

Based on GC-MS and network pharmacology, the active constituents, potential targets, and mechanism of essential oil from Gleditsiae Fructus Abnormalis(EOGFA) against cerebral ischemia/reperfusion(I/R) injury were explored, and the effective constituents were verified by experiment. To be specific, GC-MS was used identify the constituents of the volatile oil. Secondly, the targets of the constituents and disease were predicted by network pharmacology, and the drug-constituent-target network was constructed, followed by Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of the core targets. Molecular docking was performed to investigate the binding affinity between the active constituents and the targets. Finally, SD rats were used for experimental verification. The I/R injury model was established, and the neurological behavior score, infarct volume, and pathological morphology of brain tissue were measured in each group. The content of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-alpha(TNF-α) was determined by enzyme-linked immunosorbent assay(ELISA), and the protein expression of vascular endothelial growth factor(VEGF) by Western blot. A total of 22 active constituents and 17 core targets were screened out. The core targets were involved in 56 GO terms and the major KEGG pathways of TNF signaling pathway, VEGF signaling pathway, and sphingolipid signaling pathway. Molecular docking showed that the active constituents had high affinity to the targets. The results of animal experiment suggested that EOGFA can alleviate the neurological impairment, decrease the cerebral infarct volume and the content of IL-1β, IL-6 and TNF-α, and down-regulate the expression of VEGF. The experiment verified the part results of network pharmacology. This study reflects the multi-component, multi-target, and multi-pathway characteristics of EOGFA. The mechanism of its active constituents is related to TNF and VEGF pathways, which provides a new direction for in-depth research on and secondary development of Gleditsiae Fructus Abnormalis.

Mechanism of total flavonoids of Rhododendra simsii in alleviating ischemic brain injury / 中国中药杂志

This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 μg·mL~(-1)) group, and TFR(30 μg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.

Effect of Tongdu Tiaoshen electroacupuncture pretreatment on PPARγ-mediated pyroptosis of cerebral cortex in rats with cerebral ischemia reperfusion injury / 中国针灸

OBJECTIVE@#To observe the effect of Tongdu Tiaoshen (promoting the circulation of the governor vessel and regulating the spirit) electroacupuncture (EA) pretreatment on pyroptosis mediated by peroxisome proliferators-activated receptor γ (PPARγ) of the cerebral cortex in rats with cerebral ischemia reperfusion injury (CIRI) and explore the potential mechanism of EA for the prevention and treatment of CIRI.@*METHODS@#A total of 110 clean-grade male SD rats were randomly divided into a sham-operation group, a model group, an EA group, an EA + inhibitor group and an agonist group, 22 rats in each group. In the EA group, before modeling, EA was applied to "Baihui" (GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14), with disperse-dense wave, 2 Hz/5 Hz in frequency, 1 to 2 mA in intensity, lasting 20 min; once a day, consecutively for 7 days. On the base of the intervention as the EA group, on the day 7, the intraperitoneal injection with the PPARγ inhibitor, GW9662 (10 mg/kg) was delivered in the EA + inhibitor group. In the agonist group, on the day 7, the PPARγ agonist, pioglitazone hydrochloride (10 mg/kg) was injected intraperitoneally. At the end of intervention, except the sham-operation group, the modified thread embolization method was adopted to establish the right CIRI model in the rats of the other groups. Using the score of the modified neurological severity score (mNSS), the neurological defect condition of rats was evaluated. TTC staining was adopted to detect the relative cerebral infarction volume of rat, TUNEL staining was used to detect apoptosis of cerebral cortical nerve cells and the transmission electron microscope was used to observe pyroptosis of cerebral cortical neural cells. The positive expression of PPARγ and nucleotide-binding to oligomerization domain-like receptor protein 3 (NLRP3) in the cerebral cortex was detected with the immunofluorescence staining. The protein expression of PPARγ, NLRP3, cysteinyl aspartate specific protease-1 (caspase-1), gasdermin D (GSDMD) and GSDMD-N terminal (GSDMD-N) in the cerebral cortex was detected with Western blot. Using the quantitative real-time fluorescence-PCR, the mRNA expression of PPARγ, NLRP3, caspase-1 and GSDMD of the cerebral cortex was detected. The contents of interleukin (IL)-1β and IL-18 in the cerebral cortex of rats were determined by ELISA.@*RESULTS@#Compared with the sham-operation group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.01), pyroptosis was severe, the protein and mRNA expression levels of PPARγ, NLRP3, caspase-1 and GSDMD were elevated (P<0.01); and the protein expression of GSDMD-N and contents of IL-1β and IL-18 were increased (P<0.01) in the model group. When compared with the model group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1β and IL-18 were lower (P<0.01) in the EA group and the agonist group; while, in the EA + inhibitor group, the protein expression of PPARγ was increased (P<0.01), the protein and mRNA expression levels of NLRP3 and GSDMD were decreased (P<0.01, P<0.05), the mRNA expression of caspase-1 was reduced (P<0.01); and the contents of IL-1β and IL-18 were lower (P<0.01). When compared with the EA + inhibitor group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.05, P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1β and IL-18 were declined (P<0.01) in the EA group. Compared with the agonist group, in the EA group, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.05, P<0.01), the mRNA expression of PPARγ was decreased (P<0.01) and the protein expression of GSDMD-N was elevated (P<0.05); and the contents of IL-1β and IL-18 were higher (P<0.01).@*CONCLUSION@#Tongdu Tiaoshen EA pretreatment can attenuate the neurological impairment in the rats with CIRI, and the underlying mechanism is related to the up-regulation of PPARγ inducing the inhibition of NLRP3 in the cerebral cortex of rats so that pyroptosis is affected.

Effects of electroacupuncture on NLRP3/Caspase-1/GSDMD axis and neurological function in rats with cerebral ischemic reperfusion / 中国针灸

OBJECTIVE@#To investigate the neuroprotective effect of electroacupuncture (EA) at "Quchi" (LI 11) and "Zusanli" (ST 36) in the rats with cerebral ischemic reperfusion and the potential mechanism of microglia pyroptosis.@*METHODS@#Sixty SD rats were randomly divided into a sham-operation group, a model group and an EA group, with 20 rats in each group. The Zea Longa method was employed to establish the rat model of the middle cerebral artery occlusion and reperfusion (MACO/R) in the left brain. In the EA group, since the 2nd day of modeling, EA was given at "Quchi" (LI 11) and "Zusanli" (ST 36) of right side with disperse-dense wave, 4 Hz/20 Hz in frequency and 0.2 mA in current intensity, 30 min each time, once a day for lasting 7 consecutive days. The reduction rate of cerebral blood flow was measured with laser Doppler flowmetry during operation. The neurological function of rats was observed using Zea Longa neurobehavioral score. The cerebral infarction volume was detected by TTC staining method. The microglia positive expression in the ischemic side of the cortex was detected with the immunofluorescence method. Under transmission electron microscope, the ultrastructure of cell in the ischemic cortex was observed. The mRNA expression levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), cysteinyl aspartate specific proteinase-1 (Caspase-1) and gasdermin D (GSDMD) in the ischemic cortex were detected using real-time PCR.@*RESULTS@#Compared with the sham-operation group, in the model group, the reduction rate of cerebral blood flow was increased during operation (P<0.001); Zea Longa neurobehavional score and the percentage of cerebral infarction volume were increased (P<0.001), the numbers of M1-type microglia marked by CD68+ and M2-type microglia marked by TMEM119+ were elevated in the ischemic cortex (P<0.001), the mRNA expression of NLRP3, ASC, Caspase-1 and GSDMD was increased (P<0.001, P<0.01); the cytomembrane structure was destroyed, with more cell membrane pores formed in the ischemic cortex. Compared with the model group, after intervention, Zea Longa neurobehavioral score and the percentage of cerebral infarction volume were reduced (P<0.05), the number of M1-type microglia marked by CD68+ was reduced (P<0.05) and the number of M2-type microglia marked by TMEM119+ was increased (P<0.05); and the mRNA expression of NLRP3, ASC, Caspase-1 and GSDMD was decreased (P<0.01, P<0.05) in the EA group. Even though the cytomembrane structure was incomplete, there were less membrane pores presented in the ischemic cortex in the EA group after intervention.@*CONCLUSION@#The intervention with EA attenuates the neurological dysfunction and reduces the volume of cerebral infarction in the rats with cerebral ischemic reperfusion. The underlying mechanism is related to the inhibition of microglia pyroptosis through modulating NLRP3/Caspase-1/GSDMD axis.

Study on the protective effect of Hypericum perforatum on cerebral ischemia-reperfusion injury in rats and its mechanism / 中国药房

OBJECTIVE To study the protective effect and potential mechanism of Hypericum perforatum on cerebral ischemia- reperfusion in rats. METHODS The male SD rats were randomly divided into sham operation group， model group， positive control group （nimodipine 0.012 g/kg）， H. perforatum high-dose and low-dose groups （5.212， 1.303 g/kg）， with 10 rats in each group. Except for sham operation group， the left middle cerebral artery ischemia-reperfusion model was established with the modified suture method. Administration groups were given relevant medicine intragastrically since the second day after surgery， once a day， for 7 consecutive days. The neurological function scores of rats were measured before drug intervention （the first day after modeling） and after the last administration， and the cerebral infarction after the last administration was observed using TTC staining method；HE staining and TUNEL staining methods were used to observe the pathological changes of the cerebral cortex and hippocampus， and the apoptosis of nerve cells， respectively. Western blot and RT-PCR were used to observe the protein and mRNA expressions of erythropoietin （EPO）， erythropoietin receptor （EPOR）， Janus kinase 2 （JAK2） and signal transduction and activator of transcription 3 （STAT3）， and protein expression of phosphorylated STAT3 （p-STAT3）， respectively. RESULTS Compared with sham operation group， neurological function score and the proportion of cerebral infarction in model group were significantly increased before intervention and after the last administration （P＜0.01）， with significant damage to nerve cells in cerebral cortex and hippocampus， an increase in apoptotic nerve cells， and a significant increase in apoptosis rate （P＜0.01）； protein and mRNA expressions of EPO and EPOR in the brain tissue were significantly reduced （P＜0.01）， while the protein expressions of JAK2， p- STAT3 and STAT3， and mRNA expressions of JAK2 and STAT3 were significantly increased （P＜0.01）. Compared with model group， the damage and apoptosis of nerve cells in cerebral cortex and hippocampus of rats in administration groups were improved， and the quantitative indicators were almost significantly improved （P＜0.05 or P＜0.01）. CONCLUSIONS H. perforatum has a protective effect against cerebral ischemia-reperfusion injury in rats， and the related mechanism may be related to the regulation of EPO-mediated JAK2/STAT3 signaling pathway.

Research Progress on Protective Effect and Mechanism of Salidroside on Cerebral Ischemia Injury： A Review / 中国实验方剂学杂志

Ischemic stroke is caused by a variety of factors caused by intracerebral artery stenosis or obstruction， can lead to cerebral ischemia， hypoxia， neuronal necrosis and neurological dysfunction and other pathological injuries， with high morbidity， high disability rate， high mortality characteristics. Cerebral ischemia-reperfusion injury is the main secondary injury， which can lead to permanent disability or even death in severe cases. With the development of traditional Chinese medicine（TCM） modernization， the extraction and application of active components of TCM have been paid more and more attention. Salidroside， as the main active component of Rhodirosea， a rare Chinese medicinal herb， has been proved to fight cerebral ischemia injury by inhibiting cell apoptosis， anti-oxidative stress， reducing inflammatory response， protecting blood-brain barrier， regulating autophagy， promoting nerve remodeling and synaptic regeneration in preclinical trials. However， due to its multi-pathway， multi-pathway and multi-target action characteristics， the specific mechanism of salidroside to improve cerebral ischemia injury has not been fully elucidated. By reviewing relevant literature in the past decade， the author reviewed the mechanism of action of salidroside in the treatment of ischemic brain injury， and summarized the recent progress of its pharmacokinetic studies and safety evaluation， in order to provide theoretical basis and new research ideas for the development and clinical application of the active ingredients of traditional Chinese medicine.

Protective effect of Sox11 gene against cerebral ischemia reperfusion injury and its mechanism in mice / 中国生物制品学杂志

@#ObjectiveTo explore the protective effect of Sox11 gene on cerebral ischemia reperfusion injury(CIRI)in mice and its mechanism,so as to provide a new target for the treatment of CIRI.MethodsThe mouse middle cerebral artery occlusion(MCAO)model and Neuro2A cell oxygen glucose deprivation reperfusion(OGDR)model were established and detected for the temporal and spatial distribution of Sox11 in the models by real-time quantitative PCR,Western blot,immunohistochemistry(IHC)and immunohistofluorescence(IHF).The altered expression of some crucial genes in the pathway of apoptosis and inflammation in OGDR model after the disruption of Sox11 expression was detected by Western blot.ResultsThe expression level of Sox11 mRNA and protein increased significantly in both MCAO and Neuro2A OGDR models(P = 0.000 1 ～ 0.038 8);After CIRI,Sox11 expression was elevated in the hippocampal dentate gyrus(DG)region of mice;After interfering with the expression of Sox11 in OGDR model,the expression of apoptosis-related protein Cleaved Caspase 3 significantly increased,while the expression of anti-apoptosis protein Bcl-2 significantly decreased,and the expression of phosphorylated NF-κB(p-NF-κB)protein related to inflammatory reaction also up-regulated significantly.Conclusion Sox11gene had a protective effect against CIRI in mice,and was involved in the regulation of apoptosis and inflammation pathways after CIRI.

Effects of pramipexole combined with levodopa on cognitive and mitochondrial function of rats after global cerebral ischemia-reperfusion injury / 中国康复理论与实践

ObjectiveTo investigate the effects of dopamine receptor agonist pramipexole and levodopa on emotion and cognition, and mitochondrial membrane potential of rats after global cerebral ischemia-reperfusion injury. MethodsA total of 80 male Sprague-Dawley rats were divided into sham group (n = 20), model group (n = 20), pramipexole group (n = 20) and combined group (n = 20). The latter three groups were used to prepare the model of global cerebral ischemia-reperfusion injury with Pulsinelli's four-vessel occlusion. The pramipexole group was intraperitoneally injected pramipexole 0.5 mg/kg once a day, while the combined group was injected levodopa 50 mg/kg and pramipexole 0.5 mg/kg, for 14 days. Five rats in each group were tested with open field test three, seven and 14 days after modeling; five were tested with Y-maze test seven and 14 days after modeling; five were detected mitochondrial membrane potential three, seven and 14 days after modeling; and five were observed under Nissl's staining14 days after modeling. ResultsCompared with the model group, the number of entries into the central zone (P < 0.05), total distance travelled (P < 0.05) and average velocity (P < 0.05) in the open field test increased in the pramipexole and combined groups seven and 14 days after modeling, duration spent in the central zone increased in the pramipexole and combined groups seven days after modeling (P < 0.05); the rate of spontaneous alternation of Y-maze test increased in the pramipexole and combined groups 14 days after modeling (P < 0.05); mitochondrial membrane potential in hippocampus increased in the pramipexole and combined groups seven and 14 days after modeling (P < 0.05), and it was less in the pramipexole group than in the combined group 14 days after modeling (P < 0.05); and the number of surviving neurons in the hippocampal CA1 increased in the pramipexole and combined groups 14 days after modeling (P < 0.05). ConclusionPramipexole may improve emotion and cognition of rats after global cerebral ischemia-reperfusion injury, and it may be helpful for restoring mitochondrial membrane potential as combining with levodopa.

Effects of Huzhang Qingmai decoction on cognitive function and intestinal flora in mice with chronic cerebral ischemia / 药学实践杂志

Objective To investigate the mechanism of action of Huzhang Qingmai decoction (HZQMY) on the improvement of cognitive function in mice with chronic cerebral ischemia from the perspective of intestinal flora. Methods A mouse model of chronic cerebral ischemia was established by placing microcoils around the bilateral common carotid arteries to induce bilateral carotid artery stenosis (BCAS). After 12 weeks of intragastric administration, the cognitive function of the mice was measured by the Morris water maze; the myelin damage was analyzed by LFB staining; The contents of the cecum of the mice in each group were extracted and analyzed by 16S rRNA sequencing. Results The results of the water maze experiment showed that the mice in the HZQMY group had a significantly shorter escape latency, increased the number of crossings platform and the percentage of target quadrants. LFB staining showed that the white matter damage in the model group was severe; the white matter damage in the HZQMY group was milder. The results of 16S rRNA sequencing showed that compared with the model group, the abundance of Verrucomicrobiota, Akkermansia, and ErysiPelatoclostridium capsulatum in the intestinal flora in HZQMY group was significantly reduced (P<0.05), while the abundances of Eubacterium_xylanoPhilum and Allobaculum were significantly increased (P<0.05). Conclusion The protective effect of HZQMY, which has the effect of improving cognitive function in mice with chronic cerebral ischemia, may be related to the regulation of intestinal flora in mice with chronic cerebral ischemia.

Bone marrow mesenchymal stem cell-derived exosomes promote microglia/macrophage M2 polarization in acute cerebral ischemia rats and inhibit inflammatory response / 中国药科大学学报

@#The aim of the present study was to investigate the effects of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) on the polarization of M1/M2 microglia/macrophages in rats with acute cerebral ischemia.Ultrahigh-speed centrifugation was employed to isolate and identify exosomes; a middle cerebral artery occlusion (MCAO) model was prepared in rats using the intraluminal filament technique; Longa scoring and corner tests were used to evaluate the neurological function of rats; 2, 3, 5-triphenyltetrazole chloride (TTC) staining was used to assess the infarct volume in rat brains; immunofluorescence double-labeling of CD16/32/Iba1 and CD206/Iba1 was performed to detect M1/M2 phenotypes of microglia/macrophages; RT-qPCR was employed to measure the mRNA expression of CD86, inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), arginase-1 (Arg-1), interleukin-10 (IL-10), and transforming growth factor beta (TGF-β) in the ischemic penumbra of rat brains.The experimental results showed that BMSC-Exos reduced the number of CD16/32+/Iba1+ positive cells in the ischemic penumbra (P < 0.01) while increasing the number of CD206+/Iba1+positive cells (P < 0.01), and decreased the mRNA expression of iNOS, CD86, and TNF-α, while increasing the mRNA expression of Arg-1, TGF-β, and IL-10 (P < 0.05 or P < 0.01).This research suggests that BMSC-Exos can regulate M1/M2 polarization of microglia/macrophages in rats with acute cerebral ischemia, alleviate neuroinflammation, and improve ischemic brain injury.