ObjectiveTo investigate whether Huanglian Jiedutang can inhibit neutrophil infiltration in the brains of middle cerebral artery occlusion （MCAO） mice by regulating the expression of neutrophil-related chemokines in exosomes， thereby achieving therapeutic effects. MethodsA total of 130 male specific pathogen-free （SPF） C57BL/6J mice were randomly divided into four groups： Sham-operated group， MCAO model group， Huanglian Jiedutang group （6 g·kg^-1）， and Ginaton group （21.6 mg·kg^-1）， with 10 mice in the Ginaton group and 40 mice in each of the remaining three groups. Mice in the Huanglian Jiedutang group and the Ginaton group were administered the corresponding drugs by oral gavage once daily at a volume of 0.15 mL·（10 g）^-1 for 7 consecutive days， while the sham-operated and model groups received an equal volume of saline via the same route. After 7 days， MCAO surgery was performed. The distal and proximal ends of the right common carotid artery （CCA） were ligated， a small incision was made between the two ligatures， and a silicone rubber-coated monofilament with a rounded tip was inserted into the lumen to occlude the CCA. The filament was left in place for 1 h to establish a focal cerebral ischemia model. At 24 h after modeling， mice were evaluated. Neurological function was assessed using the Longa score. Cerebral infarct volume was measured by 2，3，5-triphenyltetrazolium chloride （TTC） staining. Cerebral blood flow was observed by laser speckle imaging. Hematoxylin and eosin （HE） staining and Nissl staining were used to observe pathological changes in brain tissues. Exosomes were isolated from mouse plasma and brain tissues by ultracentrifugation and molecular size exclusion and identified by electron microscopy， particle size analysis， and protein blotting. Long-chain RNA libraries of exosomes were constructed and sequenced. Real-time quantitative reverse transcription polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of inflammatory factors and neutrophil-related chemokines in exosomes from plasma and brain tissues of each group. Enzyme-linked immunosorbent assay （ELISA） was used to detect the protein expression of inflammatory factors and neutrophil-related chemokines in exosomes from brain tissues of each group. Immunohistochemistry was used to detect the expression of the neutrophil-specific protein myeloperoxidase （MPO） in the brains of mice in each group. ResultsCompared with the sham-operated group， the model group showed decreased neurological function scores （P<0.01）， obvious cerebral infarction （P<0.01）， reduced cerebral blood flow （P<0.01）， neuronal necrosis in the brain， and decreased numbers of Nissl bodies （P<0.01）. The mRNA expression levels of IL-1β， MPO， CXCL1， CXCL2， CXCL3， CXCL10， CCL2， and CCL3 in exosomes from plasma and brain tissues were significantly increased （P<0.05， P<0.01）. The protein expression levels of IL-1β， MPO， CXCL2， and CXCL10 in exosomes from brain tissues were increased （P<0.05， P<0.01）， and MPO-positive rates and mean optical density values in brain tissues were elevated （P<0.01）. Compared with the model group， the Huanglian Jiedutang group and the Ginaton group showed increased neurological function scores （P<0.05）， reduced cerebral infarct volume （P<0.01）， restored cerebral blood flow （P<0.01）， reduced necrotic cells in the brain， and increased numbers of Nissl bodies （P<0.01）. In the Huanglian Jiedutang group， the mRNA expression levels of IL-1β， MPO， CXCL1， CXCL2， CXCL3， CXCL10， CCL2， and CCL3 in exosomes from plasma and brain tissues were decreased （P<0.05， P<0.01）. The protein expression levels of IL-1β， MPO， CXCL2， and CXCL10 in exosomes from brain tissues were reduced （P<0.05， P<0.01）， and MPO-positive rates and mean optical density values in brain tissues were decreased （P<0.01）. ConclusionHuanglian Jiedutang can effectively regulate the expression of neutrophil-related chemokines in exosomes from plasma and brain tissues of MCAO mice， thereby reducing neutrophil infiltration in the brain and achieving therapeutic effects.

ObjectiveTo investigate whether Huanglian Jiedutang can inhibit neutrophil infiltration in the brains of middle cerebral artery occlusion （MCAO） mice by regulating the expression of neutrophil-related chemokines in exosomes， thereby achieving therapeutic effects. MethodsA total of 130 male specific pathogen-free （SPF） C57BL/6J mice were randomly divided into four groups： Sham-operated group， MCAO model group， Huanglian Jiedutang group （6 g·kg^-1）， and Ginaton group （21.6 mg·kg^-1）， with 10 mice in the Ginaton group and 40 mice in each of the remaining three groups. Mice in the Huanglian Jiedutang group and the Ginaton group were administered the corresponding drugs by oral gavage once daily at a volume of 0.15 mL·（10 g）^-1 for 7 consecutive days， while the sham-operated and model groups received an equal volume of saline via the same route. After 7 days， MCAO surgery was performed. The distal and proximal ends of the right common carotid artery （CCA） were ligated， a small incision was made between the two ligatures， and a silicone rubber-coated monofilament with a rounded tip was inserted into the lumen to occlude the CCA. The filament was left in place for 1 h to establish a focal cerebral ischemia model. At 24 h after modeling， mice were evaluated. Neurological function was assessed using the Longa score. Cerebral infarct volume was measured by 2，3，5-triphenyltetrazolium chloride （TTC） staining. Cerebral blood flow was observed by laser speckle imaging. Hematoxylin and eosin （HE） staining and Nissl staining were used to observe pathological changes in brain tissues. Exosomes were isolated from mouse plasma and brain tissues by ultracentrifugation and molecular size exclusion and identified by electron microscopy， particle size analysis， and protein blotting. Long-chain RNA libraries of exosomes were constructed and sequenced. Real-time quantitative reverse transcription polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of inflammatory factors and neutrophil-related chemokines in exosomes from plasma and brain tissues of each group. Enzyme-linked immunosorbent assay （ELISA） was used to detect the protein expression of inflammatory factors and neutrophil-related chemokines in exosomes from brain tissues of each group. Immunohistochemistry was used to detect the expression of the neutrophil-specific protein myeloperoxidase （MPO） in the brains of mice in each group. ResultsCompared with the sham-operated group， the model group showed decreased neurological function scores （P<0.01）， obvious cerebral infarction （P<0.01）， reduced cerebral blood flow （P<0.01）， neuronal necrosis in the brain， and decreased numbers of Nissl bodies （P<0.01）. The mRNA expression levels of IL-1β， MPO， CXCL1， CXCL2， CXCL3， CXCL10， CCL2， and CCL3 in exosomes from plasma and brain tissues were significantly increased （P<0.05， P<0.01）. The protein expression levels of IL-1β， MPO， CXCL2， and CXCL10 in exosomes from brain tissues were increased （P<0.05， P<0.01）， and MPO-positive rates and mean optical density values in brain tissues were elevated （P<0.01）. Compared with the model group， the Huanglian Jiedutang group and the Ginaton group showed increased neurological function scores （P<0.05）， reduced cerebral infarct volume （P<0.01）， restored cerebral blood flow （P<0.01）， reduced necrotic cells in the brain， and increased numbers of Nissl bodies （P<0.01）. In the Huanglian Jiedutang group， the mRNA expression levels of IL-1β， MPO， CXCL1， CXCL2， CXCL3， CXCL10， CCL2， and CCL3 in exosomes from plasma and brain tissues were decreased （P<0.05， P<0.01）. The protein expression levels of IL-1β， MPO， CXCL2， and CXCL10 in exosomes from brain tissues were reduced （P<0.05， P<0.01）， and MPO-positive rates and mean optical density values in brain tissues were decreased （P<0.01）. ConclusionHuanglian Jiedutang can effectively regulate the expression of neutrophil-related chemokines in exosomes from plasma and brain tissues of MCAO mice， thereby reducing neutrophil infiltration in the brain and achieving therapeutic effects.

Indobufen is a new generation of antiplatelet agents and has been shown to have antithrombotic effects in animal models. However, its therapeutic potential and mechanisms against cerebral ischemia/reperfusion (I/R) injury remain unclear. In this study, we evaluated the in vivo neuroprotective effects of indobufen through both pretreatment and posttreatment regimens in a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R). In vitro, human umbilical vein endothelial cells (HUVECs) subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) were employed to investigate the relationship between indobufen and the pyroptosis-associated NF-κB/Caspase-1/GSDMD pathway. The pharmacodynamic tests revealed that indobufen ameliorated I/R injury by decreasing the level of thromboxane B2 (TXB2), infarct size, brain edema and neurological impairment in rats and rescuing cell pyroptosis in HUVECs. The underlying mechanisms were probably related to pyroptosis suppression by regulating the NF-κB/Caspase-1/GSDMD pathway. Overall, these studies indicate that indobufen exerts protective and therapeutic effects against I/R injury by pyroptosis suppression via downregulating NF-κB/Caspase-1/GSDMD pathway.

AIM:To investigate autophagic status in ischemic stroke with Liver Yang Hyperactivity and the mechanism of Tianma Gouteng Decoction(TMGTD).METHODS:SD rats were divided into sham,model,TMGTD high/medium/low-dose(20.52/10.26/5.13 g·kg-1·d-1),and Nimodipine(30 mg·kg-1·d-1)groups.A Liver Yang Hyperactivity and cerebral ischemia-reperfusion model was es-tablished using Fuzi Decoction(2 g·kg-1·d-1)and thread-occlusion.After 21 days of Fuzi decoction pretreatment,rats received daily drug administra-tion for 12 days.Syndrome indicators(irritability,24-hour water intake,24-hour urine volume,facial temperature)were recorded,plasma NE,E,cAMP,and cGMP were measured by ELISA,neurological function was assessed using Zea Longa and mNSS methods,brain histopathology was evaluated by HE staining,protein expression of soluble/insoluble p62 and LC3B was detected by Western blot,au-tophagy-related genes were analyzed by PCR array,additionally,mRNA and protein levels of CXCR4 and CXCL12 were measured by qRT-PCR and Western blot.RESULTS:Compared to the sham group,the model group showed increased irritability,24-hours water intake,24-hours urine volume,facial temper-ature,and level of NE,E,cGMP(P＜0.01),neurologi-cal scores(P＜0.01),LC3B-Ⅱ,insoluble p62,CXCR4,CXCL12 expression(P＜0.01),but decreased soluble p62(P＜0.01).TMGTD groups exhibited reduced irri-tability,water intake,urine volume,facial tempera-ture,NE,E,cGMP(P＜0.05,P＜0.01),neurological scores(P＜0.05,P＜0.01),p62 expression(P＜0.01),alongside increased LC3B-Ⅱ(P＜0.01)and improved cortical pathology.TMGD also reversed dysregulat-ed autophagy-apoptosis genes(CXCR4,Lamp1,Tgfb1,APP,Rab24)and reduced CXCR4,CXCL12 ex-pression(P＜0.01).CONCLUSION:In the Liver Yang Hyperactivity and cerebral ischemia-reperfusion model,autophagy genes were activated but flux was impaired,and Tianma Gouteng Decoction may protect by restoring autophagic flux and inhibiting the CXCL12/CXCR4 axis.

Objective: To investigate the effect and mechanism of methyltransferase-like3(METTL3),which functions as m6A modification methyltransferase,on the cellular proliferation,clone formation and migration of nonsmall cell lung cancer(NSCLC) cells. Methods: The METTL3 level and patient overall survival were analyzed by bioinformatics.In the transfected A549 cells,the expression of METTL3 was detected by RT-PCR and Western blot assays,the m6A level was detected by m6A RNA Methylation Assay kit,the cellular viability and growth were detected by CCK-8 assay,the apoptosis was detected by immunofluorescent assay,the colony growth was detected by clone formation assay,the cell migration growth was detected by scratch. Results : Bioinformatics showed that METTL3 was highly expressed in NSCLC and negatively correlated with patient overall survival.The RT-PCR and Western blot data showed that the METTL3 level was higher in NSCLC cells.Moreover,higher METTL3 significantly promoted m6A level,cell proliferation,colony formation,migration,inhibited cell apoptosis,increased the mRNA and protein expressions of EMT-related marker Vimentin but inhibited apoptosis and decreased the mRNA and protein expressions of EMT-related marker E-cadherin in the A549 cells.Furthermore,the contrasting results were obtained in the A549 cells with the knockdown of METTL3. Conclusion The over-expressed METTL3 increases the m6A levels in NSCLC cells and promotes cellular proliferation,colony formation,and migration growth,thereby laying a theoretical foundation for future research on early diagnosis and targeted drug development for NSCLC by targeting METTL3.

Objective:To investigate the anti-fatigue effect of Dendrobium and Panax Quinquefolius Granules(DPQG)on the overtrained mice,and to clarify its possible mechanism.Methods:A total of 48 mice were randomly divided into control group(equal volume of distilled water),low dose of DPQG group(400 mg·kg-1 DPQG),medium dose of DPQG group(800 mg·kg-1 DPQG),and high dose of DPQG group(1 600 mg·kg-1 DPQG).The DPQG were administered by gavage for 35 d,and the rotarod test and swimming endurance test were performed 30 min after last administration.Serum,liver tissue,and muscle tissue were collected from the mice in various groups.ELISA method was used to detect the serum lacticacid(LAC)levels and lactate dehydrogenase(LDH)activities,and the malondialdehyde(MDA)levels,superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)activities,and the liver glycogen and muscle glycogen levels in muscle tissue of the mice in various groups;HE staining was used to observe the pathomorphology of muscle tissue of the mice.Transcriptomics and metabolomics technologies were used to identify the key genes and metabolites in muscle tissue of the mice in control group and high dose of DPQG group and to analyze the correlations between differentially expressed genes(DEGs)and differentially expressed metabolites.Results:Compared with control group,the rod turning exhaustion time of the mice in different doses of DPQG groups were significantly increased(P＜0.05),and the swimming exhaution time of the mice in high dose of DPQG group was increased(P＜0.05).Compared with control group,the LDH,SOD,and GSH-Px activities of the mice in medium and high doses of DPQG groups were increased(P＜0.01).Compared with control group,the levels of MDA and liver glycogen of the mice in medium and high doses of DPQG groups were decreased(P＜0.05 or P＜0.01).The transcriptomics sequencing results showed that DPQG mainly acted on DEGs such as Trib3 and Olfr495;the Gene Ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analysis results showed that the DEGs were mainly enriched in olfactory-related processes and signaling pathways;the metabolomics KEGG analysis results showed that the differential metabolites were mainly enriched in the regulation pathway of inflammatory mediators on tryptophan(TRP);the combined analysis of transcriptomics and metabolomics results showed that the piezo1 gene had high correlations with the differential metabolites β1-solamarine(r=-1,P＜0.05)and tilidine(r=1,P＜0.05).Conclusion:DPQG can exert an anti-fatigue effect on the overtrained mice by modulating LAC metabolism and glycogen homeostasis,as well as maintaining the oxidative/antioxidant balance in the body;its anti-fatigue mechanism is related to the Olfr495 and piezo1 genes and the regulation pathway of inflammatory mediators on TRP channels.

OBJECTIVE: To investigate the effects of Bushen Huoxue Granule on the ubiquitin-proteasome system (UPS) in an in vitro model of Parkinson's disease. METHODS: After treated with 1-methyl-4-phenylpyridinium (MPP⁺, 1 mmol/L) for 24 h, the cells were incubated with drug-free serum, Madopar-containing serum or Bushen Huoxue Granule-containing serum (BCS, 5%, 10%, and 20%) for another 24 h. The levels of α-synuclein (α-syn), tyrosine hydroxylase (TH) and UPS-related proteins were detected by Western blot. The expression levels of α-syn in PC12 cells were also analyzed by Western blot after treated with proteasome inhibitor MG132 and WT-α-syn plasmid transfection, respectively, as well as the alterations induced by subsequent BCS intervention. Immunocytochemistry was performed to determine the changes in α-syn phosphorylation at serine 129 (pSer129-α-syn) expression. The 20S proteasome levels were measured by enzyme-linked immunosorbnent assay. RESULTS: BCS (volume fraction ⩽20%) intervention could alleviate the MMP⁺-induced cell viability decrease (P<0.05). In the MPP⁺ treated cells, α-syn was up-regulated, while TH and proteins of UPS such as ubiquitin (Ub), Ub binding with Ub-activating enzyme (UBE1), Parkin and Ub C-terminal hydrolase-1 (UCHL-1) were down-regulated (P<0.05). BCS intervention could attenuate the above changes (P<0.05). The activity of BCS on blocking α-syn accumulation was weakened by MG132 (P<0.05). While α-syn level was significantly increased in cells transfected with plasmid, and reduced by BCS intervention (P<0.05). pSer129-α-syn was increased in MPP⁺-induced PC12 cells, whereas decreased by later BCS intervention (P<0.05). The 20S proteasome activity of MPP⁺-induced PC12 cells was decreased, but increased after BCS intervention (P<0.05). CONCLUSION BCS intervention protected UPS function, increased 20S proteasome activity, promoted pathological α-syn clearance, restored cell viability, and reversed the damage caused by MPP⁺ in the in vitro model of Parkinson's disease.
PC12 Cells ; alpha-Synuclein/metabolism* ; Rats ; Animals ; 1-Methyl-4-phenylpyridinium/toxicity* ; Proteasome Endopeptidase Complex/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Ubiquitin/metabolism* ; Cell Survival/drug effects* ; Phosphorylation/drug effects* ; Tyrosine 3-Monooxygenase/metabolism*

Objective:To investigate the clinical manifestations, long-term survival, and prognosis of childhood T-cell acute lymphoblastic leukemia (T-ALL).Methods:Case summary.The clinical data of 43 T-ALL children who were diagnosed and treated in Children′s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from January 2010 to December 2021 were retrospectively analyzed.They were stratified for treatment according to the CCCG-ALL regimen, and the correlation of prognosis with the condition at initial diagnosis, early treatment response, and induced remission was analyzed.The Kaplan-Meier survival curve was used to analyze the survival rate, and the survival rates were compared between groups by the Log-rank test.The multivariate Cox regression model was used to analyze the impact of multiple factors on the long-term survival of children.Results:T-ALL patients accounted for 9.5% (43/451) of the total number of acute lymphoblastic leukemia patients admitted to the hospital at the same period.The median onset age of the 43 T-ALL patients was 7 years (1-13 years).Of the 43 patients included, 14 patients (32.6%) had concomitant mediastinal widening, 8 patients (18.6%) had concomitant giant mediastinal masses, and 4 patients (9.3%) had early precursor T-cell acute lymphoblastic leukemia (ETP-ALL) at initial diagnosis.These 43 children were treated according to the CCCG-ALL intermediate- and high-risk group regimen.Among them, 33 children (76.7%) achieved sustained remission, 5 children died, and 5 children had a relapse.As of September 30, 2024, the median follow-up time was 62 months (1-170 months), the 10-year event-free survival rate was (80.2±6.4)%, and the 10-year overall survival rate was (86.6±5.8)%.The median relapse time and 10-year cumulative relapse rate of the 5 relapsed children were 28 months (7-58 months) and (13.7±5.8)%, respectively.The relationship of prognosis with clinical characteristics at initial diagnosis and induced remission in 43 T-ALL children was analyzed.The results showed that patients aged ≥10 years, with a grade-1 non-central nervous system at initial diagnosis, ETP-ALL, abnormal chromosome number and structure, non-M1 status of bone marrow and minimal residual disease (MRD)≥ 1% on day 19 of induction treatment, and MRD ≥ 0.01% on day 46 to 55 of induction treatment had poorer long-term survival(all P<0.05).The multivariate analysis showed that age ≥10 years, ETP-ALL, and abnormal chromosome number and structure were risk factors of poor prognosis ( P=0.045, 0.030, 0.021). Conclusions:The CCCG-ALL regimen has a good overall therapeutic effect in children with T-ALL.Age ≥10 years, abnormal chromosome number and structure, ETP-ALL, grade-1 non-central nervous system at initial diagnosis, and early remission are risk factors of poor prognosis.Treatment after relapse in children with T-ALL is difficult.