Aim To explore the protective effect of the isochroman compound Asperisochroman B(AB)on oxygen-glucose deprivation/reoxygenation(OGD/R)injury of neurons based on the PI3K/AKT/Foxo1 path-way and to reveal the related mechanism.Methods Primary neurons were cultured and the OGD/R model was constructed.The primary neurons were divided in-to the blank control group,OGD/R group,and AB low,medium,and high concentration(3,10,30 μmol·L-1)groups.The effects of AB on primary neurons were determined by CCK-8 assay,lactate dehydrogen-ase(LDH)release assay,and Hoechst 33342 stai-ning.The expression levels of PI3K,AKT,and Foxo1-related proteins were detected by Western blot.After intervention with the PI3K inhibitor(LY294002)and re-modeling and intervention with high concentra-tion of AB(30 μmol·L-1),the expression of PI3K/Foxo1 pathway-related proteins was detected by West-ern blot.Results Compared with the OGD/R group,AB could significantly increase the cell survival rate of primary neurons and reduce the release of LDH.The results of Hoechst 33342 and immunofluorescence stai-ning showed that AB reduced apoptosis after OGD/R injury.Western blot results showed that compared with the OGD/R group,after AB intervention,the expres-sion levels of Bcl-2 and NeuN proteins in neurons sig-nificantly increased(P＜0.01),and the expression level of Bax protein significantly decreased(P＜0.01).At the same time,it upregulated the expres-sion levels of p-AKT and PI3K proteins,promoted Foxo1 phosphorylation,and downregulated the expres-sion of Foxo1.Compared with the high-dose AB group,LY294002 could inhibit the changes of the a-bove indicators and reverse the protective effect of AB on OGD/R-injured primary neurons.Conclusions AB can alleviate oxygen-glucose deprivation/reoxygen-ation-induced neuronal injury,and its mechanism may be related to the activation of the PI3K/AKT/Foxo1 signaling pathway.

Aim To investigate the protective effect of Gualou Guizhi granules(GLGZG)on neuronal injury induced by LPS-activated microglia based on Notch signaling pathway.Methods LPS-activated microglia were co-cultured with neurons to construct neuron inju-ry models,and the cells were divided into the control group,model group,Notch inhibitor(DAPT)group,GLGZG(50,100,200 mg·L-1)group,DAPT+100 mg·L-1GLGZG group.After intervention,the activity of HT22 cells was detected by CCK-8 method,and rel-ative mRNA expression was detected by real-time PCR.The relative protein expression was detected by Western blot.Results Compared with the model group,after GLGZG intervention,the cell activity was significantly improved,GLGZG decreased IL-6,IL-12,Bax,Notch 1,caspase-3,Delta-1,NICD,RBPSUH,HES1 expression,and increased Bcl-2 expression(P＜0.05).Compared with the model group,the NICD,RBPSUH and HES1 mRNA and protein expressions significantly decreased after DAPT treatment(P＜0.05),and there was no superposition effect with GLG-ZG.Conclusion GLGZG may play a neuroprotective role by inhibiting inflammatory factors and apoptosis,and inhibiting Notch signaling pathway.

Objective:To investigate the effect of baicalin on Th17/Treg cell balance in autoimmune myocarditis(AM)rats and its possible mechanism.Methods:AM rats were grouped into control group,model group,low-dose baicalin group[20 mg/(kg·d)],high-dose baicalin group[100 mg/(kg·d)],and high mobility group B1(HMGB1)inhibitor group;the cardiac ultrasound diagnostic instrument was applied to detect the cardiac function of rats in each group,the cardiac mass index,hematoxylin eosin(HE)staining was applied to detect myocardial pathology,ELISA was applied to detect serum levels of cytokines such as IFN-γ,IL-4,IL-17 and TGF-β,Western blot was applied to detect the expression levels of myocardial HMGB1 and receptor for advanced glycation end product(RAGE)proteins,flow cytometry was applied to detect the proportions of Th17 and Treg cells in the spleen.Results:Com-pared with the control group,the rats in the model group showed myocarditis symptoms and diffuse inflammatory cell infiltration under the endocardium,the left ventricular end diastolic diameter(LVEDd),left ventricular end systolic diameter(LVEDs),IFN-γ,IL-4,IL-17,TGF-β,the proportion of Th17 cells and the expression levels of HMGB1 and RAGE proteins increased,the left ventricular short axis shortening fraction(FS%),ejection fraction(LVEF%),cardiac mass index,and the proportion of Treg cell decreased(P<0.05);compared with the model group,the myocardial interstitial edema and inflammatory cell infiltration were reduced in the ba-icalin low and high dose groups and HMGB1 inhibitor groups,and the proportion of LVEDd,LVEDs,serum IFN-γ and IL-17,spleen Th17 cells and the expression levels of myocardial HMGB1 and RAGE protein were decreased.FS%,LVEF%,heart mass index,IL-4,TGF-β,Treg cell proportion increased;compared with the low-dose baicalin group,the symptoms of myocarditis in the high-dose baicalin group and the HMGB1 inhibitor group were obviously improved,the LVEDd,LVEDs,IFN-γ,IL-17,the proportion of Th17 cells,and the expression levels of HMGB1 and RAGE proteins decreased,the FS%,LVEF%,cardiac mass index,IL-4,TGF-β,and the proportion of Treg cells increased(P<0.05);there was no statistically obvious difference in each index between the high-dose baicalin group and the HMGB1 inhibitor group(P>0.05).Conclusion:The protective effect of baicalin on AM rats is related to the inhibition of HMGB1/RAGE axis activation.

Aim To investigate the mechanism of Guanxinning injection regulating cardiac immune mi-croenvironment to improve myocardial infarction in mice.Methods In this study,MI model was estab-lished by permanent ligation of left anterior descending coronary artery in mice.The mice were divided into five groups:sham operation group,model group,Guanxinning injection low dose group,Guanxinning in-jection high dose group and positive drug captopril group.Hearts were weighed,heart tissues were collect-ed,and Masson staining was used for pathological anal-ysis of heart tissues;immunofluorescence staining was used to detect apoptosis and CCL21 expression in the infarct border zone;flow cytometry was used to detect the proportion of immune cells in myocardial ischemia tissues and lymph nodes;PCR was used to detect CCL21 expression in heart and in vitro human lymphat-ic endothelial cells(HLEC).Results Compared with the model group,the low and high dose groups of Guanxinning injection significantly improved cardiac hypertrophy.Apoptosis in the border zone of myocardi-al infarction was reduced in the low and high dose groups of Guanxinning injection and captopril group.Compared with the model group,the proportion of leu-kocytes in the infarct border zone was dreduced and the proportion of CD4+T cells,Treg cells,and CD8+T cells in the mediastinal lymph nodes and infarct border zone of the heart was regulated in the low and high dose groups of Guanxinning injection;CCL21 secretion by the heart and lymphatic vessels increased.Conclu-sions Guanxinning injection can significantly improve cardiac hypertrophy and fibrosis in MI mice,reduce ap-optosis in the infarct border zone,and play a role in an-ti-myocardial ischemia injury by promoting CCL21 ex-pression in lymphatic vessels to regulate the proportion of mediastinal lymph nodes and cardiac T cells after myocardial infarction.

Aim To investigate the mechanism of Guanxinning injection regulating cardiac immune mi-croenvironment to improve myocardial infarction in mice.Methods In this study,MI model was estab-lished by permanent ligation of left anterior descending coronary artery in mice.The mice were divided into five groups:sham operation group,model group,Guanxinning injection low dose group,Guanxinning in-jection high dose group and positive drug captopril group.Hearts were weighed,heart tissues were collect-ed,and Masson staining was used for pathological anal-ysis of heart tissues;immunofluorescence staining was used to detect apoptosis and CCL21 expression in the infarct border zone;flow cytometry was used to detect the proportion of immune cells in myocardial ischemia tissues and lymph nodes;PCR was used to detect CCL21 expression in heart and in vitro human lymphat-ic endothelial cells(HLEC).Results Compared with the model group,the low and high dose groups of Guanxinning injection significantly improved cardiac hypertrophy.Apoptosis in the border zone of myocardi-al infarction was reduced in the low and high dose groups of Guanxinning injection and captopril group.Compared with the model group,the proportion of leu-kocytes in the infarct border zone was dreduced and the proportion of CD4+T cells,Treg cells,and CD8+T cells in the mediastinal lymph nodes and infarct border zone of the heart was regulated in the low and high dose groups of Guanxinning injection;CCL21 secretion by the heart and lymphatic vessels increased.Conclu-sions Guanxinning injection can significantly improve cardiac hypertrophy and fibrosis in MI mice,reduce ap-optosis in the infarct border zone,and play a role in an-ti-myocardial ischemia injury by promoting CCL21 ex-pression in lymphatic vessels to regulate the proportion of mediastinal lymph nodes and cardiac T cells after myocardial infarction.

Aim To investigate the protective effect of Gualou Guizhi granules(GLGZG)on neuronal injury induced by LPS-activated microglia based on Notch signaling pathway.Methods LPS-activated microglia were co-cultured with neurons to construct neuron inju-ry models,and the cells were divided into the control group,model group,Notch inhibitor(DAPT)group,GLGZG(50,100,200 mg·L-1)group,DAPT+100 mg·L-1GLGZG group.After intervention,the activity of HT22 cells was detected by CCK-8 method,and rel-ative mRNA expression was detected by real-time PCR.The relative protein expression was detected by Western blot.Results Compared with the model group,after GLGZG intervention,the cell activity was significantly improved,GLGZG decreased IL-6,IL-12,Bax,Notch 1,caspase-3,Delta-1,NICD,RBPSUH,HES1 expression,and increased Bcl-2 expression(P＜0.05).Compared with the model group,the NICD,RBPSUH and HES1 mRNA and protein expressions significantly decreased after DAPT treatment(P＜0.05),and there was no superposition effect with GLG-ZG.Conclusion GLGZG may play a neuroprotective role by inhibiting inflammatory factors and apoptosis,and inhibiting Notch signaling pathway.

Objective To investigate the effect of different residence time of povidone iodine on the disinfection effect of conjunctival sac in senile cataract surgery.Methods A total of 396 senile patients who underwent cataract surgery in our hospital were selected and randomly divided into the long-residence group and the short-residence group,with 198 cases in each group.The patients of the long-residence group and the short-residence group were disinfected with a disposable filling 5%povidone iodine on conjunctival sac for 3 minutes and 30 seconds before surgery,respectively.The bacterial detection and bacterial distribution on conjunctival sac,and corneal epithelial cell damage were compared between the two groups.The subjective comfort conditions such as ocular itching,foreign body sensation,erythema,and photophobia after disinfection were recorded.Results There was no significant difference in the bacterial detection rate before and after disinfection between the two groups(P>0.05).The most frequently detected bacteria in the short-residence group was Staphylococcus epidermidis,accounting for 28.79%and 33.33%before and after disinfection,respectively.The incidences of ocular itching,foreign body sensation,erythema,and photophobia after disinfection in the short-residence group were significantly lower than those in the long-residence group(P<0.05),while the rate of corneal epithelial cell damage in the long-residence group was significantly higher than that in the short-residence group(P<0.05).Conclusion The use of disposable filling 5%povidone iodine for disinfection on conjunctival sac for 30 seconds before surgery can achieve a comparable disinfection effect to disinfection for 3 minutes,which has less effect on corneal epithelial cells,and higher ocular comfort.

Objective:To investigate the effect of baicalin on Th17/Treg cell balance in autoimmune myocarditis(AM)rats and its possible mechanism.Methods:AM rats were grouped into control group,model group,low-dose baicalin group[20 mg/(kg·d)],high-dose baicalin group[100 mg/(kg·d)],and high mobility group B1(HMGB1)inhibitor group;the cardiac ultrasound diagnostic instrument was applied to detect the cardiac function of rats in each group,the cardiac mass index,hematoxylin eosin(HE)staining was applied to detect myocardial pathology,ELISA was applied to detect serum levels of cytokines such as IFN-γ,IL-4,IL-17 and TGF-β,Western blot was applied to detect the expression levels of myocardial HMGB1 and receptor for advanced glycation end product(RAGE)proteins,flow cytometry was applied to detect the proportions of Th17 and Treg cells in the spleen.Results:Com-pared with the control group,the rats in the model group showed myocarditis symptoms and diffuse inflammatory cell infiltration under the endocardium,the left ventricular end diastolic diameter(LVEDd),left ventricular end systolic diameter(LVEDs),IFN-γ,IL-4,IL-17,TGF-β,the proportion of Th17 cells and the expression levels of HMGB1 and RAGE proteins increased,the left ventricular short axis shortening fraction(FS%),ejection fraction(LVEF%),cardiac mass index,and the proportion of Treg cell decreased(P<0.05);compared with the model group,the myocardial interstitial edema and inflammatory cell infiltration were reduced in the ba-icalin low and high dose groups and HMGB1 inhibitor groups,and the proportion of LVEDd,LVEDs,serum IFN-γ and IL-17,spleen Th17 cells and the expression levels of myocardial HMGB1 and RAGE protein were decreased.FS%,LVEF%,heart mass index,IL-4,TGF-β,Treg cell proportion increased;compared with the low-dose baicalin group,the symptoms of myocarditis in the high-dose baicalin group and the HMGB1 inhibitor group were obviously improved,the LVEDd,LVEDs,IFN-γ,IL-17,the proportion of Th17 cells,and the expression levels of HMGB1 and RAGE proteins decreased,the FS%,LVEF%,cardiac mass index,IL-4,TGF-β,and the proportion of Treg cells increased(P<0.05);there was no statistically obvious difference in each index between the high-dose baicalin group and the HMGB1 inhibitor group(P>0.05).Conclusion:The protective effect of baicalin on AM rats is related to the inhibition of HMGB1/RAGE axis activation.

The study aimed to investigate the effect of the CD200R1 gene deletion on microglia activation and nigrostriatal dopamine neuron loss in the Parkinson's disease (PD) process. The CRISPR-Cas9 technology was applied to construct the CD200R1^-/- mice. The primary microglia cells of wild-type and CD200R1^-/- mice were cultured and treated with bacterial lipopolysaccharide (LPS). Microglia phagocytosis level was assessed by a fluorescent microsphere phagocytosis assay. PD mouse model was prepared by nigral stereotaxic injection of recombinant adeno-associated virus vector carrying human α-synuclein (α-syn). The changes in the motor behavior of the mice with both genotypes were evaluated by cylinder test, open field test, and rotarod test. Immunohistochemical staining was used to assess the loss of dopamine neurons in substantia nigra. Immunofluorescence staining was used to detect the expression level of CD68 (a key molecule involved in phagocytosis) in microglia. The results showed that CD200R1 deletion markedly enhanced LPS-induced phagocytosis in vitro by the microglial cells. In the mouse model of PD, CD200R1 deletion exacerbated motor behavior impairment and dopamine neuron loss in substantia nigra. Fluorescence intensity analysis results revealed a significant increase in CD68 expression in microglia located in the substantia nigra of CD200R1^-/- mice. The above results suggest that CD200R1 deletion may further activates microglia by promoting microglial phagocytosis, leading to increased loss of the nigrostriatal dopamine neurons in the PD model mice. Therefore, targeting CD200R1 could potentially serve as a novel therapeutic target for the treatment of early-stage PD.
Animals ; Microglia/physiology* ; Mice ; Phagocytosis ; Parkinson Disease/genetics* ; Disease Models, Animal ; Receptors, Cell Surface/physiology* ; Dopaminergic Neurons/pathology* ; Antigens, CD/metabolism* ; Gene Deletion ; Substantia Nigra ; Mice, Inbred C57BL ; Mice, Knockout ; Cells, Cultured ; Male ; alpha-Synuclein ; CD68 Molecule ; Orexin Receptors

The study aimed to construct the second and third generation chimeric antigen receptor T cells (CAR-T) targeting the c-mesenchymal-epithelial transition factor (c-Met) protein, and observe their killing effect on human serous ovarian cancer cell SKOV-3. The expression of MET gene in ovarian serous cystadenocarcinoma, the correlation between MET gene expression and the abundance of immune cell infiltration, and the effect of MET gene expression on the tissue function of ovarian serous cystadenocarcinoma were analyzed by bioinformatics. The expression of c-Met in ovarian cancer tissues and adjacent tissues was detected by immunohistochemical staining. The second and third generation c-Met CAR-T cells, namely c-Met CAR-T(2G/3G), were prepared by lentivirus infection, and the cell subsets and infection efficiency were detected by flow cytometry. Using CD19 CAR-T and activated T cells as control groups and A2780 cells with c-Met negative expression as Non target groups, the kill efficiency on SKOV-3 cells with c-Met positive expression, cytokine release and cell proliferation of c-Met CAR-T(2G/3G) were explored by lactate dehydrogenase (LDH) release, ELISA and CCK-8 respectively. The results showed that MET gene expression was significantly up-regulated in ovarian cancer tissues compared with normal tissues, which was consistent with the immunohistochemistry results. However, in all pathological stages, there was no obvious difference in MET expression and no correlation between MET gene expression and the race and age of ovarian cancer patients. The second generation and third generation c-Met CAR-T cells were successfully constructed. After lentivirus infection, the proportion of CD8⁺ T cells in c-Met CAR-T(2G) was upregulated, while there was no significant change in the cell subsets of c-Met CAR-T(3G). The LDH release experiment showed that the kill efficiency of c-Met CAR-T(2G/3G) on SKOV-3 increased with the increase of effect-target ratio. When the effect-target ratio was 20:1, the kill efficiency of c-Met CAR-T(2G) reached (42.02 ± 5.17)% (P < 0.05), and the kill efficiency of c-Met CAR-T(3G) reached (51.40 ± 2.71)% (P < 0.05). ELISA results showed that c-Met CAR-T released more cytokine compared to CD19 CAR-T and activated T cells (P < 0.05). Moreover, the cytokine release of c-Met CAR-T(3G) was higher than c-Met CAR-T(2G) (P < 0.01). The CCK-8 results showed that after 48 h, the cell number of c-Met CAR-T(2G) was higher than that of c-Met CAR-T(3G) (P < 0.01). In conclusion, both the second and third generation c-Met CAR-T can target and kill c-Met-positive SKOV-3 cells, with no significant difference. c-Met CAR-T(2G) has stronger proliferative ability, and c-Met CAR-T(3G) releases more cytokines.
Humans ; Female ; Ovarian Neoplasms/immunology* ; Proto-Oncogene Proteins c-met/metabolism* ; Receptors, Chimeric Antigen/immunology* ; Cell Line, Tumor ; Cystadenocarcinoma, Serous/immunology* ; T-Lymphocytes/immunology*