Objective To investigate the protective effect of Notch1-regulated microglial polarization against epileptic seizure in mice.Methods The pentylenetetrazole(PTZ)kindling models of epilepsy were established in mice with normal(Notch1normal)and high(Notch1over)expression of the Notch1 protein,with Racine grade Ⅳ set as the standard,to evaluate the effect of Notch1 expression on sei-zure susceptibility.Microglial activation and polarization in the temporal lobe tissues of mice were detected using immunofluorescence staining,and the levels of inflammatory cytokines in the temporal lobe tissues was detected using an enzyme-linked immunosorbent assay.Results High Notch1 expression showed high resistance to seizures.Compared with the Notch1normal+Sal group,the relative fluorescence intensity of Iba1,CD16,and Arg1 proteins in the temporal lobes of mice in the Notch1normal+PTZ group,as well as the TNF-α,IL-6,and IL-10 levels significantly increased(P＜0.05).Compared with the Notch1nornal+PTZ group,the relative fluorescence intensity of Iba1 and CD16 proteins in the temporal lobes of mice in the Notch1over+PTZ group significantly decreased(P＜0.05),the relative fluorescence intensity of Arg1 protein significantly increased(P＜0.05),the TNF-α and IL-6 levels significantly decreased(P＜0.05),and the IL-10 level further increased(P＜0.05).Conclusion High Notch1 protein expression significantly enhanced M2-type microglial polari-zation,inhibited M1-type microglial polarization,and increased resistance to seizure susceptibility in mice.

Objective To systematically investigate biomechanical characteristics of one-finger Zen push method applied at the Fengchi acupoint under different force conditions,in order to provide precise quantitative data and enhance the treatment efficacy.Methods Ten senior Tuina practitioners were recruited.The German Novel Pliance-X 32 Expert dynamic pressure distribution system was used to record the mechanical parameters during the application of one-finger Zen push at the Fengchi acupoint under three force intensities:light,medium,and heavy,for 3 minutes.Data from the stable 1-minute segment of the mechanical output was selected for data analysis,and key biomechanical parameters such as the maximum force,average force,peak pressure,mean pressure,force-time integral(FTI),pressure-time integral(PTI),and operational frequency were evaluated.Results Under light,medium,and heavy force conditions,the mean maximum force applied by senior Tuina practitioners at the Fengchi acupoint were 6.31,9.45,and 18.27 N,respectively,while the mean force were 3.31,5.64,and 9.05 N,respectively.The mean peak pressures were 26.10,34.80,and 70.00 kPa,while the mean pressures were 11.95,21.00,and 26.15 kPa,respectively.The mean FTIs were 55.65,182.10,and 225.21 N·s,and the mean PTIs were 167.10,489.59,and 795.83 kPa·s,respectively.The mean operational frequencies were 156.00,150.60,and 154.80 times/min,respectively.Conclusions Ten senior Tuina practitioners showed a high degree of consistency between their subjective definitions of light,medium,and heavy force and the objectively measured mechanical parameters.This reflected their precise control over the applied force under different force conditions,verifying the practicality and reproducibility of the one-finger Zen push method in clinical applications.This study provides a reliable basis for quantitative research and development of standardized clinical operation guidelines.

Objective To systematically investigate biomechanical characteristics of one-finger Zen push method applied at the Fengchi acupoint under different force conditions,in order to provide precise quantitative data and enhance the treatment efficacy.Methods Ten senior Tuina practitioners were recruited.The German Novel Pliance-X 32 Expert dynamic pressure distribution system was used to record the mechanical parameters during the application of one-finger Zen push at the Fengchi acupoint under three force intensities:light,medium,and heavy,for 3 minutes.Data from the stable 1-minute segment of the mechanical output was selected for data analysis,and key biomechanical parameters such as the maximum force,average force,peak pressure,mean pressure,force-time integral(FTI),pressure-time integral(PTI),and operational frequency were evaluated.Results Under light,medium,and heavy force conditions,the mean maximum force applied by senior Tuina practitioners at the Fengchi acupoint were 6.31,9.45,and 18.27 N,respectively,while the mean force were 3.31,5.64,and 9.05 N,respectively.The mean peak pressures were 26.10,34.80,and 70.00 kPa,while the mean pressures were 11.95,21.00,and 26.15 kPa,respectively.The mean FTIs were 55.65,182.10,and 225.21 N·s,and the mean PTIs were 167.10,489.59,and 795.83 kPa·s,respectively.The mean operational frequencies were 156.00,150.60,and 154.80 times/min,respectively.Conclusions Ten senior Tuina practitioners showed a high degree of consistency between their subjective definitions of light,medium,and heavy force and the objectively measured mechanical parameters.This reflected their precise control over the applied force under different force conditions,verifying the practicality and reproducibility of the one-finger Zen push method in clinical applications.This study provides a reliable basis for quantitative research and development of standardized clinical operation guidelines.

Objective To investigate the protective effect of Notch1-regulated microglial polarization against epileptic seizure in mice.Methods The pentylenetetrazole(PTZ)kindling models of epilepsy were established in mice with normal(Notch1normal)and high(Notch1over)expression of the Notch1 protein,with Racine grade Ⅳ set as the standard,to evaluate the effect of Notch1 expression on sei-zure susceptibility.Microglial activation and polarization in the temporal lobe tissues of mice were detected using immunofluorescence staining,and the levels of inflammatory cytokines in the temporal lobe tissues was detected using an enzyme-linked immunosorbent assay.Results High Notch1 expression showed high resistance to seizures.Compared with the Notch1normal+Sal group,the relative fluorescence intensity of Iba1,CD16,and Arg1 proteins in the temporal lobes of mice in the Notch1normal+PTZ group,as well as the TNF-α,IL-6,and IL-10 levels significantly increased(P＜0.05).Compared with the Notch1nornal+PTZ group,the relative fluorescence intensity of Iba1 and CD16 proteins in the temporal lobes of mice in the Notch1over+PTZ group significantly decreased(P＜0.05),the relative fluorescence intensity of Arg1 protein significantly increased(P＜0.05),the TNF-α and IL-6 levels significantly decreased(P＜0.05),and the IL-10 level further increased(P＜0.05).Conclusion High Notch1 protein expression significantly enhanced M2-type microglial polari-zation,inhibited M1-type microglial polarization,and increased resistance to seizure susceptibility in mice.

OBJECTIVE To evaluate the effictiveness and safety of recombinant human endostatin combined with afatinib and teggio in the treatment of advanced lung squamous cell carcinoma. METHODS A total of 25 patients with driver-negative advanced lung squamous cell carcinoma were included in this single-arm prospective study, and the enrolled patients were treated with recombinant human endostatin combined with afatinib and teggio as scheduled. Progression-free survival(PFS), overall survival(OS), disease control rate(DCR), objective response rate(ORR), and adverse reactions(AR) were observed and analyzed. RESULTS The 25 enrolled patients received at least 2 cycles of second-line treatment, and were followed up as of March 31, 2023. Among them, 4 patients had partial remission, 17 patients had stable disease, and 4 patients experienced progressive disease. The ORR confirmed by the researchers was 16%(95%CI, 4.5%−36.1%), DCR was 84%(95%CI, 63.9%−95.5%), and median PFS was 5.3 months(95%CI, 3.5−6.9 months). The median OS had not yet been achieved. The entire group of patients had good treatment tolerance, and the most common level Ⅲ or Ⅳ adverse events related to treatment were leukopenia(20%) and rash(12%), with no reported treatment-related deaths. CONCLUSION Recombinant human endostatin combined with afatinib and teggio in the second line treatment of advanced lung squamous cell carcinoma can prolong the progression free survival period of patients and is relatively safe, which is worth further exploration and promotion.

Objective:To investigate the mechanism by which glycoprotein non-transferable melanin B(GPNMB)regulates microglia M2 polarization to reduce nerve damage after cerebral ischemic stroke(CIS).Methods:SD rats were used for establishment of middle cerebral artery occlusion(MCAO)model.Neurons,astrocytes and microglia were cultured under oxygen and glucose depri-vation(OGD)conditions,and the expression of GPNMB in tissues and cells were measured by Western blot.Gpnmb wrapped with adeno-associated virus(AAV)or shRNA-Gpnmb were injected into rat brain tissues for overexpression or inhibition of GPNMB,modified neurological deficit score(mNSS),Rotarod fatigue test and tape removal test were used to evaluate rat nerve function,the proportion of cerebral infarction was determined by TTC staining,microglia M1/M2 polarization markers were detected by immunofluorescence and RT-PCR,and the expression of Phosphatidylinositol 3-kinase(PI3K)/Serine/threonine kinase(Akt)pathway was determined by Western blot.Microglia was cultured under OGD conditions in vitro,Gpnmb was overexpressed and PI3K expression were inhibited by LY294002,and M1/M2 polarization markers were measured.Results:Compared with normal rats or normal cultured cells,the expres-sion of GPNMB in MCAO model or OGD-intervened microglia was up-regulated(P<0.05);when Gpnmb was overexpressed in the brain tissue of MCAO rats,the mNSS score decreased,the Rotarod time of latency to fall lengthened,the contact time and removal time shortened in the tape removal test,the proportion of cerebral infarction decreased,the M1 polarization level of microglia decreased while the M2 polarization level increased,PI3K/Akt pathway activated,and these difference were statistically significant(P<0.05);inhibition of PI3K reversed the effect of overexpression of Gpnmb on promoting M2 polarization of microglia in vitro(P<0.05).Conclusion:GPNMB promotes M2 polarization of microglia by activating the PI3K/Akt pathway,thereby reducing nerve damage after CIS.

OBJECTIVE To investigate the effect and mechanism of Poria cocos polysaccharides on the regulation of blood glucose in type 2 diabetes mellitus （T2DM）model rats by phosphatidylinositol 3-kinase（PI3K）/protein kinase B （Akt）/forked box transcription factor O 1（FoxO1）pathway. METHODS SD rats were randomly divided into blank control group （no modeling ，no administration），model group （modeling，no administration ），metformin group （modeling，200 mg/kg）and P. cocos polysaccharide low-dose，medium-dose and high-dose groups （modeling，100，200，400 mg/kg），8 in each group. Except for blank control group ， other groups were given high fat diet combined with streptozotocin to construct the model of T 2DM rats. At the same time ， administration groups were given relevant dose of medicine intragastrically ，and blank control group and model group were given constant volume of water intragastrically ，once a day ，for consecutive 42 days. During the experiment ，general condition and bodyweight of rats were observed every day ；fasting blood glucose （FBG）of rats were collected ，and oral glucose tolerance test were conducted and area under curve （AUC）was calculated the day before last administration. After last medication ，the heart ，liver， kidney organ index were calculated ；the levels of HbA 1c，TC，TG，MDA，SOD，GSH-Px and hepatic glycogen content were detected. HE staining was used to observe the pathological changes of liver and pancreatic tissue ，and the pathological grade score was calculated. Western blot assay was used to detect the protein expressions of p-PI 3K，p-Akt，p-FoxO1， PEPCK and G 6Pase in liver tissues. RESULTS Compared with blank control group ，the rats of model group suffered cc1965@163.com from polydipsia ，polyphagia and polyuria ；the body weight ， the levels of SOD and GSH-Px ，the protein expressions of p-PI 3K，p-Akt and p-FoxO 1 were significantly decreased （P＜0.05）；liver and kidney organ index ，blood glucose level at 0，0.5 and 2 hours after intragastric administration of glucose solution ，AUC， FBG，HbA1c，serum levels of MDA ，TC，TG and hepatic glycogen content ，liver and pancreatic pathological grade score ，the protein expressions of PEPCK and G 6Pase were all increased significantly （P＜0.05）. Compared with model group ，the general condition of rats in P. cocos polysaccharide groups were all improved ，and all of above indicators had been reversed to varying degrees. CONCLUSIONS P. cocos polysaccharide can downregulate protein expressions of PEPCK and G 6Pase which are key enzymes of gluconeogenesis ，inhibit hepatic gluconeogenesis ，effectively decrease blood glucose levels and regulate glucolipid metabolism in T 2DM model rats by weakening oxidative stress and upregulating PI 3K/Akt/FoxO1 pathway.

Objective To observe the changes of corresponding proteins and function based on known clinical dihydroorotate dehydrogenase(DHODH) mutation types,i.e.,G202A,R346W and R135C in the patients with Miller syndrome.Methods HeLacell lines stably expressing Miller syndrome pathogenic mutation types G202A,R346W and R135C were established.Then the mitochondrial localization function,protein stability and enzyme activity of corresponding proteins were studied by the immunohistochemistry and mitochondrial layered positioning.Results The mitochondrial localization function of 3 kinds of DHODH mutation were not affected,which existed in the mitochondrial inner membrane after expression.The mutant G202A and R346W protein stability was reduced;the mutant R135C protein was stable,but base induced enzyme activity injury caused the deficiency of corresponding enzymatic activity.Conclusion The DHODH function injury may be related with the symptoms in Miller syndrome.

Objective To observe the changes of the skeletal development related cells after dihydroorotate dehydrogenase (DHODH) deficiency.Methods The DHODH expression in MC3T3-E1 cells derived from mouse calvaria osteoblast precursor cells was inhibited by specific small interfering RNAs (siRNAs),and cell proliferation,ATP production and expression levels of bone-related genes were investigated in these cells.Results After reducing the DHODH expression by using specific siRNAs,cell proliferation was inhibited and cell cycle was arrested at G1/S stage.In addition,the ATP production was reduced in whole cells,especially in mitochondria.Furthermore,the expression levels of Runt-related transcription factor 2 (Runx2) and osteocalcin (Ocn) mRNAs in the DHODH inhibition group were decreased compared with the control group.Conclusion Inhibiting DHODH protein affects the differentiation and maturation of osteoblasts.The mitochondrial dysfunction in osteoblasts may be one of causes leading to the abnormal bone formation in Miller syndrome.

Objective To investigate the T cell cytotoxicity induced by recombinant adenovirus carrying HIV-1 vpr gene.Methods C8166 cells infected with rAd-vpr or negative control rAd-vector,were analyzed for cell cycle distribution and cell death by flow cytometry.The discrimination of living cells,apoptotic and necrotic cells were differentiated with Hoechst-PI double staining under the confocal microscopy.Changes of mitochondrial membrane potential(△ψm)were monitored by JC-1 staining method.Results Annexin V-PI and Hoechst-PI staining indicated the death effects of HIV-1 Vpr on C8166 cells.PI flow cytometric analysis showed that cell cycle arrested in G2 phase.C8166 cell△ψm collapse mediated by Vpr was detected by JC-1 fluorescent staining.Conclusion The ability of recombinant adenovirus carrying HIV-1 vpr gene to induce mitochondria dysfunction,cell cycle G2 phase arrest and cell death was confirmed in C8166 cells.