AIM:This study aims to investigate the protective effect of Buyang-Huanwu decoction(BYHWD)on cerebral ischemia-reperfusion injury(CIRI)in rats,focusing on its role in regulating the autophagy of cerebral micro-vascular endothelial cells(BMECs).METHODS:(1)We established a rat model of middle cerebral artery occlusion/re-perfusion(MCAO/R)and divided the subjects into four groups:sham group,model(MCAO/R)group,BYHWD group,and 3-n-butylphthalide(NBP)group.Neurological deficits were assessed using the Zea Longa score,while the volume of cerebral infarction was measured through 2,3,5-triphenyltetrazolium chloride(TTC)staining.Pathological damage in the ischemic penumbra was evaluated using HE staining,and blood-brain barrier(BBB)permeability was assessed by Evans blue(EB)staining.The ultrastructure of BMECs was analyzed by transmission electron microscopy,and the co-expres-sion and positive cell rate of microtubule-associated protein 1 light chain 3(LC3)in BMECs were determined through im-munofluorescence double staining.Additionally,the protein expression levels of ZO-1,claudin-5 and occludin in the cor-tical region of the ischemic penumbra in rats were examined using Western blot analysis.(2)A rat BMEC model of oxy-gen-glucose deprivation/reoxygenation(OGD/R)was also established.Rat BMECs were categorized into normal control(CON),OGD/R,dimethyl sulfoxide(DMSO),rapamycin and 3-methyladenine groups to observe autophagy levels by monodansylcadaverine(MDC)staining.Furthermore,rat BMECs were divided into CON,OGD/R,BYHWD-containing serum(BHDS)and NBP groups.The cell autophagy was assessed by MDC staining and Western blot,while cell viability was measured by CCK-8 assay.RESULTS:(1)The rats in MCAO/R group exhibited significantly higher neurological scores(P＜0.01)and increased cerebral infarction volumes(P＜0.01)compared with sham group.Severe damage in the ischemic penumbra was observed,characterized by disordered tissue structure,widened intercellular spaces,and compro-mised cellular integrity.The EB dye permeability was notably elevated(P＜0.01),and BMECs showed structural destruc-tion,including damaged cell membranes,swollen Golgi apparatus,dilated endoplasmic reticulum vesicles,and damaged mitochondria.The ratio of LC3+CD31+/CD31+and the protein levels of ZO-1,claudin-5 and occludin were significantly el-evated(P＜0.01).In contrast,the rats in BYHWD and NBP groups demonstrated lower neurological scores(P＜0.01)and reduced cerebral infarction volumes(P＜0.01).Furthermore,EB permeability decreased(P＜0.01),BMEC morphol-ogy improved,and the protein expression levels of ZO-1,claudin-5 and occludin increased(P＜0.05).(2)Rat BMECs in OGD/R group had a significantly elevated autophagy level compared with CON group(P＜0.01),with increased expres-sion of LC3 and beclin-1 proteins and decreased level of P62 protein(P＜0.05).Notably,the cells in BHDS and NBP groups displayed decreased autophagy level compared with OGD/R group,with increased cell viability(P＜0.01),re-duced LC3 and beclin-1 protein expression,and increased P62 protein expression(P＜0.05).CONCLUSION:Buyang-Huanwu decoction alleviates cerebral ischemia-reperfusion injury in rats by inhibiting the autophagy of cerebral microvas-cular endothelial cells.

AIM:To investigate the mechanism by which exosomes(EXOs)derived from neural stem cells(NSCs)pretreated with astragaloside IV(ASIV)alleviate brain damage in rats after ischemic stroke.METHODS:Rat NSCs were isolated from fetal rats within 24 h of birth,cultured for 3 d,and subsequently treated with ASIV for additional 5 d.The EXOs from untreated NSCs and ASIV-pretreated NSCs(ASIV-EXOs)were isolated via ultracentrifugation of the cell supernatant.These EXOs were characterized using Western blot to detect specific markers such as CD63,tumor sus-ceptibility gene 101(TSG101)and calnexin.Nanoparticle analysis was employed to determine the size,and the morpholo-gy of the EXOs was observed under electron microscope.Six to eight-week-old SD male rats were randomly assigned to 6 groups:sham group,middle cerebral artery occlusion/reperfusion(MCAO/R)model group,edaravone(EDA)treatment(MCAO/R+EDA)group,EXOs treatement(MCAO/R+EXOs)group and ASIV-EXOs treatment(MCAO/R+ASIV-EXOs)group.Tail vein injections were administered within 2 h following the successful establishment of the MCAO/R model.The Zea Longa method was utilized to evaluate neurological deficits,while the TTC method was employed to assess brain infarc-tion.Pathological changes were examined through HE staining,and TUNEL and caspase-1 immunofluorescence double staining were conducted to detect cellular pyroptosis.Serum levels of interleukin-1β(IL-1β)and IL-18 were measured us-ing ELISA,and Western blot was performed to evaluate the expression of caspase-1,nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),gasdermin D(GSDMD),and IL-18 proteins in the ischemic area of the rat cerebral cortex across all groups.RE-SULTS:The MCAO/R group exhibited significantly higher neurological deficit scores compared to the sham group(P＜0.01)and lower scores in the administered groups relative to the MCAO/R group(P＜0.05).Cerebral infarction was mark-edly increased in the MCAO/R group compared to the sham group(P＜0.01),whereas the infarction area was reduced in the administered groups compared to the MCAO/R group(P＜0.05).Serum levels of IL-1β and IL-18 were significantly el-evated in the MCAO/R group versus the sham group(P＜0.01)and were lower in the administered groups compared to the MCAO/R group(P＜0.01).Moreover,IL-1β and IL-18 levels in the MCAO/R+ASIV-EXOs group were lower than those in the MCAO/R+EXOs group(P＜0.05).HE staining revealed pronounced sieve-like infarction foci in the ischemic area of the rat cerebral cortex in MCAO/R group,characterized by disorganized neuronal arrangements,reduced or absent Nys-trom's vesicles,shrunken or fragmented nuclei,and numerous red neurons.In contrast,drug-treated groups exhibited milder pathological changes with clearer neuronal structures and a significant reduction in red neuron counts.Immunofluo-rescence double staining indicated a significant increase in double-positive cells in the MCAO/R group compared to the sham group(P＜0.01),with a decrease in double-positive cells in the administered groups relative to the MCAO/R group(P＜0.05)and a further reduction in the MCAO/R+ASIV-EXOs group compared to the MCAO/R+EXOs group(P＜0.05).The expression levels of caspase-1,NLRP3,ASC,IL-18 and GSDMD proteins in the ischemic region of the rat cerebral cortex were significantly reduced in the administered groups compared to the MCAO/R group(P＜0.01),with further re-duction observed in the MCAO/R+ASIV-EXOs group compared to the MCAO/R+EXOs group(P＜0.05).CONCLU-SION:Exosomes derived from ASIV-pretreated NSCs attenuate brain damage in ischemic stroke rats,potentially through a mechanism involving the inhibition of pyroptosis mediated by the NLRP3/caspase-1 pathway.

AIM:This study aims to investigate the protective effect of Buyang-Huanwu decoction(BYHWD)on cerebral ischemia-reperfusion injury(CIRI)in rats,focusing on its role in regulating the autophagy of cerebral micro-vascular endothelial cells(BMECs).METHODS:(1)We established a rat model of middle cerebral artery occlusion/re-perfusion(MCAO/R)and divided the subjects into four groups:sham group,model(MCAO/R)group,BYHWD group,and 3-n-butylphthalide(NBP)group.Neurological deficits were assessed using the Zea Longa score,while the volume of cerebral infarction was measured through 2,3,5-triphenyltetrazolium chloride(TTC)staining.Pathological damage in the ischemic penumbra was evaluated using HE staining,and blood-brain barrier(BBB)permeability was assessed by Evans blue(EB)staining.The ultrastructure of BMECs was analyzed by transmission electron microscopy,and the co-expres-sion and positive cell rate of microtubule-associated protein 1 light chain 3(LC3)in BMECs were determined through im-munofluorescence double staining.Additionally,the protein expression levels of ZO-1,claudin-5 and occludin in the cor-tical region of the ischemic penumbra in rats were examined using Western blot analysis.(2)A rat BMEC model of oxy-gen-glucose deprivation/reoxygenation(OGD/R)was also established.Rat BMECs were categorized into normal control(CON),OGD/R,dimethyl sulfoxide(DMSO),rapamycin and 3-methyladenine groups to observe autophagy levels by monodansylcadaverine(MDC)staining.Furthermore,rat BMECs were divided into CON,OGD/R,BYHWD-containing serum(BHDS)and NBP groups.The cell autophagy was assessed by MDC staining and Western blot,while cell viability was measured by CCK-8 assay.RESULTS:(1)The rats in MCAO/R group exhibited significantly higher neurological scores(P＜0.01)and increased cerebral infarction volumes(P＜0.01)compared with sham group.Severe damage in the ischemic penumbra was observed,characterized by disordered tissue structure,widened intercellular spaces,and compro-mised cellular integrity.The EB dye permeability was notably elevated(P＜0.01),and BMECs showed structural destruc-tion,including damaged cell membranes,swollen Golgi apparatus,dilated endoplasmic reticulum vesicles,and damaged mitochondria.The ratio of LC3+CD31+/CD31+and the protein levels of ZO-1,claudin-5 and occludin were significantly el-evated(P＜0.01).In contrast,the rats in BYHWD and NBP groups demonstrated lower neurological scores(P＜0.01)and reduced cerebral infarction volumes(P＜0.01).Furthermore,EB permeability decreased(P＜0.01),BMEC morphol-ogy improved,and the protein expression levels of ZO-1,claudin-5 and occludin increased(P＜0.05).(2)Rat BMECs in OGD/R group had a significantly elevated autophagy level compared with CON group(P＜0.01),with increased expres-sion of LC3 and beclin-1 proteins and decreased level of P62 protein(P＜0.05).Notably,the cells in BHDS and NBP groups displayed decreased autophagy level compared with OGD/R group,with increased cell viability(P＜0.01),re-duced LC3 and beclin-1 protein expression,and increased P62 protein expression(P＜0.05).CONCLUSION:Buyang-Huanwu decoction alleviates cerebral ischemia-reperfusion injury in rats by inhibiting the autophagy of cerebral microvas-cular endothelial cells.

AIM:To investigate the mechanism by which exosomes(EXOs)derived from neural stem cells(NSCs)pretreated with astragaloside IV(ASIV)alleviate brain damage in rats after ischemic stroke.METHODS:Rat NSCs were isolated from fetal rats within 24 h of birth,cultured for 3 d,and subsequently treated with ASIV for additional 5 d.The EXOs from untreated NSCs and ASIV-pretreated NSCs(ASIV-EXOs)were isolated via ultracentrifugation of the cell supernatant.These EXOs were characterized using Western blot to detect specific markers such as CD63,tumor sus-ceptibility gene 101(TSG101)and calnexin.Nanoparticle analysis was employed to determine the size,and the morpholo-gy of the EXOs was observed under electron microscope.Six to eight-week-old SD male rats were randomly assigned to 6 groups:sham group,middle cerebral artery occlusion/reperfusion(MCAO/R)model group,edaravone(EDA)treatment(MCAO/R+EDA)group,EXOs treatement(MCAO/R+EXOs)group and ASIV-EXOs treatment(MCAO/R+ASIV-EXOs)group.Tail vein injections were administered within 2 h following the successful establishment of the MCAO/R model.The Zea Longa method was utilized to evaluate neurological deficits,while the TTC method was employed to assess brain infarc-tion.Pathological changes were examined through HE staining,and TUNEL and caspase-1 immunofluorescence double staining were conducted to detect cellular pyroptosis.Serum levels of interleukin-1β(IL-1β)and IL-18 were measured us-ing ELISA,and Western blot was performed to evaluate the expression of caspase-1,nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),gasdermin D(GSDMD),and IL-18 proteins in the ischemic area of the rat cerebral cortex across all groups.RE-SULTS:The MCAO/R group exhibited significantly higher neurological deficit scores compared to the sham group(P＜0.01)and lower scores in the administered groups relative to the MCAO/R group(P＜0.05).Cerebral infarction was mark-edly increased in the MCAO/R group compared to the sham group(P＜0.01),whereas the infarction area was reduced in the administered groups compared to the MCAO/R group(P＜0.05).Serum levels of IL-1β and IL-18 were significantly el-evated in the MCAO/R group versus the sham group(P＜0.01)and were lower in the administered groups compared to the MCAO/R group(P＜0.01).Moreover,IL-1β and IL-18 levels in the MCAO/R+ASIV-EXOs group were lower than those in the MCAO/R+EXOs group(P＜0.05).HE staining revealed pronounced sieve-like infarction foci in the ischemic area of the rat cerebral cortex in MCAO/R group,characterized by disorganized neuronal arrangements,reduced or absent Nys-trom's vesicles,shrunken or fragmented nuclei,and numerous red neurons.In contrast,drug-treated groups exhibited milder pathological changes with clearer neuronal structures and a significant reduction in red neuron counts.Immunofluo-rescence double staining indicated a significant increase in double-positive cells in the MCAO/R group compared to the sham group(P＜0.01),with a decrease in double-positive cells in the administered groups relative to the MCAO/R group(P＜0.05)and a further reduction in the MCAO/R+ASIV-EXOs group compared to the MCAO/R+EXOs group(P＜0.05).The expression levels of caspase-1,NLRP3,ASC,IL-18 and GSDMD proteins in the ischemic region of the rat cerebral cortex were significantly reduced in the administered groups compared to the MCAO/R group(P＜0.01),with further re-duction observed in the MCAO/R+ASIV-EXOs group compared to the MCAO/R+EXOs group(P＜0.05).CONCLU-SION:Exosomes derived from ASIV-pretreated NSCs attenuate brain damage in ischemic stroke rats,potentially through a mechanism involving the inhibition of pyroptosis mediated by the NLRP3/caspase-1 pathway.

AIM:This study aimed to investigate the effects of sinomenine hydrochloride(SIN)on the ultra-structure of renal tissue and the expression of interferon gene-stimulating factor in db/db mice.METHODS:Sixteen 12-week-old male db/db mice were randomly divided into two groups:a model group and a sinomenine hydrochloride(SIN)group,each consisting of 8 mice.An additional 8 wild-type(WT)mice served as the normal control group.The sinome-nine hydrochloride group was administered the treatment for 8 weeks,followed by a 20-week observation period,while the normal and model groups received an equal volume of saline via gavage.Weekly measurements were taken for body weight and fasting blood glucose.Serum creatinine(SCr)and blood urea nitrogen(BUN)levels were assessed,and 24-hour uri-nary microalbumin(ALB)levels,as well as serum inflammatory cytokines interleukin-1β(IL-1β),IL-6 and tumor necro-sis factor-α(TNF-α),were determined using ELISA.Pathological changes in renal tissue were evaluated through hema-toxylin-eosin(HE)staining,while ultrastructural alterations were examined using transmission electron microscopy.Im-munohistochemistry and Western blotting were employed to assess STING protein expression in renal tissue,and STING mRNA expression was quantified via RT-qPCR.RESULTS:Compared to the normal group,the model group exhibited significant increases in BUN,ALB,and SCr levels(P<0.01),alongside elevated inflammatory markers IL-1β,IL-6,and TNF-α(P<0.01).Notable pathological changes included leukocyte wall thickening in capillaries,inflammatory cell infiltration,increased mesangial matrix,disorganized and linear alignment of podocytes,and thickening of the basement membrane.Moreover,STING protein and mRNA expression levels were significantly elevated(P<0.01).In contrast,the sinomenine hydrochloride group demonstrated significantly reduced levels of renal function markers(BUN,ALB and SCr)compared to the model group(P<0.01),as well as decreased concentrations of inflammatory factors IL-1β,IL-6,and TNF-α(P<0.01).Improvements in renal histopathology included decreased leukocyte wall thickening,reduced inflam-matory cell presence,diminished mesangial matrix,and a significant reduction in foot process fusion,alongside thinner basement membranes.Both STING protein and mRNA expression levels were also significantly lower(P<0.01).CON-CLUSION:Sinomenine hydrochloride effectively mitigates renal tissue injury,improves ultrastructural alterations,and inhibits inflammatory responses in db/db mice.Its mechanism of action appears closely linked to the downregulation of STING protein and mRNA expression.