BACKGROUND:Ammonia poisoning is considered to be the main hypothesis for the pathogenesis of hepatic encephalopathy.Ammonia can lead to psychiatric and cognitive behavioral disorders,although the specific pathological molecular mechanisms remain unclear.OBJECTIVE:To investigate the effects of ammonia poisoning on cognitive behavior and hippocampal neuronal synapses in mice.METHODS:Thirty-two C57BL/6J mice were randomly divided into a normal control group and an ammonium chloride group,with 16 mice in each group.Normal saline was injected intraperitoneally in the control group,and ammonium chloride(10 mmol/kg)was injected intraperitoneally in the ammonium chloride group to construct a model of ammonia poisoning,once a day.After 7 days of ammonium chloride intervention,blood samples were collected from the hearts of six mice in each group for blood ammonia concentration detection.Behavioral experiments,including the open field test,novel object recognition test,and Y-maze test,were performed to assess mental and cognitive-behavioral changes in mice.Finally,hippocampal tissues were extracted for western blot analysis to detect the expression levels of synaptophysin and postsynaptic density protein-95 in hippocampal neurons.RESULTS AND CONCLUSION:The blood ammonia concentration was significantly elevated in the ammonium chloride group compared with the control group(P＜0.05).Mice in the ammonium chloride group showed anxiety-like behavior and disinhibition phenomenon,and a significant decrease in recognition memory and working memory ability.Western blot results revealed that the expression of synaptophysin and postsynaptic density protein-95 protein in hippocampal neurons in the ammonium chloride group was lower than that in the control group(P＜0.05).To conclude,ammonia poisoning can induce hippocampal neuronal synaptic damage,leading to psychiatric and cognitive behavioral abnormalities in mice.

BACKGROUND:Ammonia poisoning is considered to be the main hypothesis for the pathogenesis of hepatic encephalopathy.Ammonia can lead to psychiatric and cognitive behavioral disorders,although the specific pathological molecular mechanisms remain unclear.OBJECTIVE:To investigate the effects of ammonia poisoning on cognitive behavior and hippocampal neuronal synapses in mice.METHODS:Thirty-two C57BL/6J mice were randomly divided into a normal control group and an ammonium chloride group,with 16 mice in each group.Normal saline was injected intraperitoneally in the control group,and ammonium chloride(10 mmol/kg)was injected intraperitoneally in the ammonium chloride group to construct a model of ammonia poisoning,once a day.After 7 days of ammonium chloride intervention,blood samples were collected from the hearts of six mice in each group for blood ammonia concentration detection.Behavioral experiments,including the open field test,novel object recognition test,and Y-maze test,were performed to assess mental and cognitive-behavioral changes in mice.Finally,hippocampal tissues were extracted for western blot analysis to detect the expression levels of synaptophysin and postsynaptic density protein-95 in hippocampal neurons.RESULTS AND CONCLUSION:The blood ammonia concentration was significantly elevated in the ammonium chloride group compared with the control group(P＜0.05).Mice in the ammonium chloride group showed anxiety-like behavior and disinhibition phenomenon,and a significant decrease in recognition memory and working memory ability.Western blot results revealed that the expression of synaptophysin and postsynaptic density protein-95 protein in hippocampal neurons in the ammonium chloride group was lower than that in the control group(P＜0.05).To conclude,ammonia poisoning can induce hippocampal neuronal synaptic damage,leading to psychiatric and cognitive behavioral abnormalities in mice.

AIM:To observe the neuroprotective effects of total saponins of Trillium tschonoskii Maxim(TST)on rats with vascular dementia(VD)and explore the drug's impact on astrocytes and the Sonic Hedgehog(Shh)pathway as well as its mechanisms of action.METHODS:A rat model of vascular dementia was established by bilateral common carotid artery occlusion.Subsequently,the rats were randomly divided into four groups:donepezil hydrochloride group,TST group,model group,and sham group,with 18 rats in each group.After 5 weeks of gavage,samples were collected.Cognitive function was evaluated by the water maze test.Histopathological changes in brain tissue were examined with HE and Nissl staining.The ultrastructure of astrocytes was analyzed by transmission electron microscopy.The localization and expression of the neuronal nuclear antigen(NeuN),glial fibrillary acidic protein(GFAP),Shh,and glioma-associated oncogene homolog 1(Gli1)were identified using immunohistochemistry.Immunofluorescence was also employed to exam-ine the expression of NeuN and GFAP.Finally,Western blot analysis was used to measure the protein levels of NeuN,GFAP,Shh,Patched 1(Ptch1),Gli1,Bax,Bcl-2,tumor necrosis factor-α(TNF-α),and interleukin-10(IL-10)in the hippocampus.RESULTS:Compared to the sham group,the model rats demonstrated prolonged escape latency,disorga-nized and loosened neuronal arrangement in the hippocampus and cortex,reduced Nissl bodies,and significant neuronal damage.Astrocytes displayed chromatin aggregation,swollen mitochondria with disrupted cristae structures,and swollen endoplasmic reticulum regions.The expression of NeuN,Shh,and Gli1 positive cells significantly decreased,while GFAP positive cells significantly increased.Additionally,the protein levels of NeuN,Shh,Ptch1,Gli1,IL-10,and Bcl-2 in the hippocampus were reduced(P＜0.05),whereas those of GFAP,Bax,and TNF-α were elevated(P＜0.01).Com-pared to the model group,the donepezil hydrochloride and TST groups effectively improved the aforementioned indicators.CONCLUSION:Total saponins of trillium tschonoskii maxim can effectively alleviate pathological damage to neurons in VD rats,thereby improving learning and memory abilities.The underlying mechanisms may involve inhibiting the overacti-vation of astrocytes,activate the Shh/Ptch1/Gli1 signaling pathway,suppress neuroinflammation,and reduce neuronal apoptosis.

Getah virus(GETV)is an arbovirus capable of infecting humans and vertebrates such as horses and pigs via blood-sucking mosquitoes,which is extremely harmful to the livestock indus-try.Current monitoring methods are time-consuming,costly,and dependent on specialized instru-ments.These characteristics hinder rapid detection in clinical samples.Therefore,the development of a simple,rapid,specific,and sensitive method for detecting GETV antigen is crucial for the pre-vention and control of GETV.In this study,a GETV E1 monoclonal antibody strain SD17/09-E1-mAb was prepared by a prokaryotic expression system for GETV E1 protein expression,and a col-loidal gold double-antibody sandwich assay encapsulating two strains of GETV E1 monoclonal an-tibody wasestablished.The results showed that the prepared colloidal gold test strips had good sen-sitivity and did not cross-react with other common porcine virus-positive tissue samples;the test strips had a high compliance rate with the IFA assay for GETV,and could be stored at 37 ℃ for one month and at room temperature for at least three months.In this study,a colloidal gold anti-surveillance test strip for rapid detection of GETV was successfully prepared,which provides a powerful tool for GETV detection.

Getah virus(GETV)is an arbovirus capable of infecting humans and vertebrates such as horses and pigs via blood-sucking mosquitoes,which is extremely harmful to the livestock indus-try.Current monitoring methods are time-consuming,costly,and dependent on specialized instru-ments.These characteristics hinder rapid detection in clinical samples.Therefore,the development of a simple,rapid,specific,and sensitive method for detecting GETV antigen is crucial for the pre-vention and control of GETV.In this study,a GETV E1 monoclonal antibody strain SD17/09-E1-mAb was prepared by a prokaryotic expression system for GETV E1 protein expression,and a col-loidal gold double-antibody sandwich assay encapsulating two strains of GETV E1 monoclonal an-tibody wasestablished.The results showed that the prepared colloidal gold test strips had good sen-sitivity and did not cross-react with other common porcine virus-positive tissue samples;the test strips had a high compliance rate with the IFA assay for GETV,and could be stored at 37 ℃ for one month and at room temperature for at least three months.In this study,a colloidal gold anti-surveillance test strip for rapid detection of GETV was successfully prepared,which provides a powerful tool for GETV detection.

AIM:To observe the neuroprotective effects of total saponins of Trillium tschonoskii Maxim(TST)on rats with vascular dementia(VD)and explore the drug's impact on astrocytes and the Sonic Hedgehog(Shh)pathway as well as its mechanisms of action.METHODS:A rat model of vascular dementia was established by bilateral common carotid artery occlusion.Subsequently,the rats were randomly divided into four groups:donepezil hydrochloride group,TST group,model group,and sham group,with 18 rats in each group.After 5 weeks of gavage,samples were collected.Cognitive function was evaluated by the water maze test.Histopathological changes in brain tissue were examined with HE and Nissl staining.The ultrastructure of astrocytes was analyzed by transmission electron microscopy.The localization and expression of the neuronal nuclear antigen(NeuN),glial fibrillary acidic protein(GFAP),Shh,and glioma-associated oncogene homolog 1(Gli1)were identified using immunohistochemistry.Immunofluorescence was also employed to exam-ine the expression of NeuN and GFAP.Finally,Western blot analysis was used to measure the protein levels of NeuN,GFAP,Shh,Patched 1(Ptch1),Gli1,Bax,Bcl-2,tumor necrosis factor-α(TNF-α),and interleukin-10(IL-10)in the hippocampus.RESULTS:Compared to the sham group,the model rats demonstrated prolonged escape latency,disorga-nized and loosened neuronal arrangement in the hippocampus and cortex,reduced Nissl bodies,and significant neuronal damage.Astrocytes displayed chromatin aggregation,swollen mitochondria with disrupted cristae structures,and swollen endoplasmic reticulum regions.The expression of NeuN,Shh,and Gli1 positive cells significantly decreased,while GFAP positive cells significantly increased.Additionally,the protein levels of NeuN,Shh,Ptch1,Gli1,IL-10,and Bcl-2 in the hippocampus were reduced(P＜0.05),whereas those of GFAP,Bax,and TNF-α were elevated(P＜0.01).Com-pared to the model group,the donepezil hydrochloride and TST groups effectively improved the aforementioned indicators.CONCLUSION:Total saponins of trillium tschonoskii maxim can effectively alleviate pathological damage to neurons in VD rats,thereby improving learning and memory abilities.The underlying mechanisms may involve inhibiting the overacti-vation of astrocytes,activate the Shh/Ptch1/Gli1 signaling pathway,suppress neuroinflammation,and reduce neuronal apoptosis.

Lung cancer is one of the most common cancers worldwide, and its mortality rate remains high. In addition to conventional surgery, radiotherapy, and chemotherapy, immunotherapy methods have been developed and used in recent years for the treatment of non-small cell lung cancer (NSCLC). However, only a small number of patients with NSCLC can benefit from immunotherapy strategies, and some patients even have hyperprogression after receiving immunotherapy. Therefore, precision immunotherapy requires effective biomarkers to guide it. In this paper, tissue samples, blood samples, intestinal microbiota, and other biomarkers are reviewed according to different sample sources. Blood samples, including TCR immune repertoire, Tregs cells, cytokines, lactate dehydrogenase, and other markers, are summarized and analyzed to provide reference for clinicians' diagnosis and treatment decisions.

Objective:To discuss the effect of concentrated growth factor(CGF)on the performance of bone marrow mesenchymal stem cells(BMSCs)sheets,and to clarify the role of CGF-containing composite cell sheets(CS)in the bone defect repairment.Methods:In in vitro experiments,the BMSCs were isolated and cultured from two 3-week-old SD rats;Alizarin Red S and Oil Red O staining were used to identify the osteogenic and adipogenic capabilities of BMSCs;CGF liquid extracts(CGFe)was prepared from three 3-week-old SD rats.The cells were divided into control group,traditional CS(BMSC-CS)group,and CGF-containing composite CS(CGF/BMSC-CS)group.The morphology of the CS in two groups was observed by HE staining.Alizarin Red and alkaline phosphatase(ALP)staining were used to detect the osteogenic differentiation of the CS in various groups;cell scratch assay was used to detect the migration abilities of the cells in various groups;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the mRNA expression levels of ALP,collagen are type 1(COL-1),Runt-related transcription factor 2(RUNX2),and osteocalcin(OCN)in the cells in various groups.In in vivo experiments,15 SD rats were randomly divided into control group,BMSC-CS group,and CGF/BMSC-CS group;micro computed tomography(Micro-CT)was used to detect the bone formation parameters in skull defects of the rats in various groups;HE staining and Masson staining were used to observe the morphology of skull defect tissue of the rats in various groups.Results:The third-generation BMSCs were spindle-shaped,closely arranged,and grew in a vortex cluster.The Alizarin red staining results showed obvious calcium nodules,and the Oil red O staining showed red lipid droplets,confirming the cells'ability to undergo osteogenic and adipogenic differentiation.The CS were white and semi-transparent,with slightly curled edges.The peeled CS were irregularly curled and wrinkled.Compared with BMSC-CS group,the CS in CGF/BMSC-CS group were whiter,less transparent,significantly increased in thickness and extensibility,less prone to breakage,and had a certain degree of stickiness and plasticity.The HE staining results showed that compared with BMSC-CS group,the number of the cells of CS in CGF/BMSC-CS group was increased,with denser arrangement and more abundant extracellular matrix(ECM),which wrapped and connected the cells to form an integral sheet-like structure.The Alizarin red and ALP staining results showed that compared with control group,the ALP activity and mineralization uplift value of CS in BMSC-CS group were significantly increased(P<0.05);compared with control group and BMSC-CS group,the number of osteoblasts and red mineralized nodules in the CS in CGF/BMSC-CS group was significantly increased,with obvious deepening of the staining,increased positive area,and the ALP activity and mineralization uplift value were significantly increased(P<0.05).Compared with BMSC-CS group,the ALP activity and mineralization uplif value of the CS in CGF/DMSC-CS group were increased(P<0.05).The cell scratch assay results showed that after 24 of culture,compared with control group,the migration rates of the cells in BMSC-CS group and CGF/BMSC-CS group were significantly increased(P<0.05).Compared with BMSC-CS group,the migration rate of the cells in CGF/BMSC-CS group was significantly increased(P<0.01).After 48 h of culture,compared with control group,the migration rate of the cells in CGF/BMSC-CS group was significantly increased(P<0.05).The RT-qPCR results showed that compared with control group,the expression levels of COL-1 and OCN mRNA in the cells in BMSC-CS group were significantly increased(P<0.01),and the expression levels of ALP,COL-1,OCN,and RUNX2 mRNA in the cells in CGF/BMSC-CS group were significantly increased(P<0.01).Compared with BMSC-CS group,the expression levels of ALP,COL-1,and OCN mRNA in the cells in CGF/BMSC-CS group were significantly increased(P<0.01).The Micro-CT detection results showed that in control group,the boundary of the rat skull defect area was clear,with almost no new bone formation.In BMSC-CS group,a small amount of new bone formed only at the edge of the bone defect in skull of the rats,with a significant gap in the central area of the defect.In CGF/BMSC-CS group,new bone formed along the edge of the bone defect towards the central area in skull of the rats,repairing most of the bone defect.Compared with control group,the bone volume(BV)and trabecular number(Tb.N)of the rats in BMSC-CS group were significantly increased(P<0.05);the bone volume(BV),bone volume fraction[BV/tissue volume(TV)],trabecular thickness(Tb.Th),and trabecular number(Tb.N)in skull of the rats in CGF/BMSC-CS group,were significantly increased(P<0.05).Compared with BMSC-CS group,the BV,BV/TV,Tb.Th,and Tb.N in skull of the rats in CGF/BMSC-CS group were significantly increased(P<0.01).The HE and Masson staining observation showed that in control group,almost no new bone formed in the skull defect tissue of the rats,with only a large amount of collagen fibers connecting the two sides of the bone ends.In BMSC-CS group,a small amount of new bone formed only at the edge of the bone defect in skull tissue of the rats,with the central area of the defect containing dense collagen fibers connected to the newly formed bone at the defect edge.In CGF/BMSC-CS group,new bone tissue could be seen at the edge of the bone defect,and bone islands formed in the central area of the defect,surrounded by osteocytes and a large amount of collagen fibers.The Masson staining observation results showed that the cytoplasm and osteoid were red,and the collagen was blue.In CGF/BMSC-CS group,newly formed osteoid was observed in skull defect tissue of the rats,with the highest amount of new bone formation.Conclusion:CGF can promote the osteogenic differentiation and increase the richness of ECM in BMSCs sheets.CGF-containing composite CS can efficiently repair skull defects of the rats and serve as an ideal and safe material for promoting the bone regeneration.

Objective:To discuss the therapeutic effect of resveratrol on the temporomandibular joint osteoarthritis(TMJOA),and to clarify the related mechanism.Methods:Forty-five SD rats were randomly divided into control group,model group,and resveratrol group,and there were 15 rats in each group.The rats in model group and resveratrol group were intra-articularly injected with 50 μL of 20 g·L-1 monosodium iodoacetate(MIA)to set TMJOA rat models,while the rats in control group were injected with an equal volume of normal saline.Three weeks after modeling,the rats in resveratrol group received an injection of 80 μL resveratrol solution,once a week for three weeks,while the rats in control and model groups were injected with an equal volume of normal saline.Micro-computed tomography(Micro-CT)system was used to detect the condyle structure and the bone volume fraction(BV/TV),trabecular thickness(Tb.Th),trabecular spacing(Tb.Sp),and trabecular number(Tb.N)of the rats in various groups were calculated;HE staining and toluidine blue staining were used to observe the pathomorphology of temporomandibular joint(TMJ)tissue of the rats in various groups;immunohistochemistry was used to detect the expression levels of SRY-related HMG box(SOX)-9,matrix metalloproteinase(MMP)-13,silent information regulator(Sirt)1,phosphatidylinositol 3-kinase(PI3K),phosphorylated protein kinase B(p-Akt),and phosphorylated mammalian target of rapamycin(p-mTOR)in TMJ tissue of the rats in various groups;real-time quantitative PCR(RT-qPCR)method was used to detect the expression levels of SOX-9,MMP-13,Sirt1,PI3K,mTOR,and Akt mRNA in TMJ tissue of the rats in various groups.Results:Three weeks after modeling,condylar bone was destructed,the surface was roughness,and continuity interruption were observed,indicating TMJOA model of the rats was established successfully.The Micro-CT system results showed that the condylar surface of the rats in control group was smooth and regularly shaped,with continuous bone texture;the rats in model group had significant condylar destruction,disrupted continuity,surface roughness,and varying degrees of bone defects;the rats in resveratrol group showed alleviated condylar lesions and improved appearance.Compared with control group,the BV/TV and Tb.Th of the rats in model group were significantly decreased(P<0.05),and Tb.Sp was significantly increased(P<0.05);compared with model group,the BV/TV and Tb.Th of the rats in resveratrol group were significantly increased(P<0.05),and the Tb.Sp was significantly decreased(P<0.05).The HE staining results showed clear layers and orderly chondrocyte arrangement in condyle of the rats in control group;the rats in model group showed rough uneven surface,obvious defects,and typical TMJOA features;the rats in resveratrol group showed slightly rough surface with generally clear layers and orderly arranged cells.The toluidine blue staining results showed distinct blue-purple staining of chondrocytes in hypertrophic layer of the rats in control group;pale staining or even loss of staining in some areas of the rats in model group;and distinct and relatively uniform staining in hypertrophic layer of the rats in resveratrol group.The immunohistochemistry results showed that compared with control group,the expression levels of MMP-13,PI3K,p-Akt,and p-mTOR proteins in TMJ tissue of the rats in model group were significantly increased(P<0.05),while the expression levels of SOX-9 and Sirt1 proteins in TMJ tissue of the rats were significantly decreased(P<0.05);compared with model group,the expression levels of SOX-9 and Sirt1 proteins in TMJ tissue of the rats in resveratrol group were significantly increased(P<0.05),whereas the expression levels of MMP-13,PI3K,p-Akt,and p-mTOR proteins were significantly decreased(P<0.05).The RT-qPCR results showed that compared with control group,the expression levels of MMP-13,PI3K,Akt,and mTOR mRNA in TMJ tissue of the rats in model group were significantly increased(P<0.05),while the expression levels of SOX-9 and Sirt1 mRNA were significantly decreased(P<0.05);compared with model group,the expression levels of SOX-9 and Sirt1 mRNA in TMJ tissue of the rats in resveratrol group were significantly increased(P<0.05),whereas the expression levels of MMP-13,PI3K,Akt,and mTOR mRNA were significantly decreased(P<0.05).Conclusion:Resveratrol has therapeutic effect on TMJOA,and its mechanism may be related to the activation of Sirt1 and inhibition of the PI3K-Akt-mTOR signaling pathway.

Objective To investigate the effect of long non-coding RNA(lncRNA)lung cancer-related transcript 1(LUCAT1)on the pathogenesis of intrauterine adhesion by regulating the expression of amphiregulin(AREG),to provide a new molecular target for the prevention and treatment of intrauterine adhesions.Methods Real-time quantitative polymerase chain reaction(RTqPCR)was used to determine the mRNA expression levels of lncRNA LUCAT1,fibrotic markers of α-smooth muscle actin(α-SMA)and collagen type Ⅰ alpha 1 chain protein(COL1A1)in samples of intrauterine adhesion tissue and endometrial stromal cell treated with transforming growth factor-β1.Western blot was used to determine the protein expression levels of AREG,α-SMA,COL1A1.Then,si-LUCAT1,pcDNA LUCAT1,si-AREG were transfected into ESC and RT-qPCR was used to detect the mRNA expression levels of lncRNA LUC-AT1 and AREG.Next,si-LUCAT1 and pcDNA LUCAT1 were transfected into ESC and treated these cells with TGF-β1 for 48h,re-spectively.Western blot was used to further detect the protein expression levels of AREG,α-SMA and COL1A1,and cell proliferation and apoptosis were detected by cell proliferation assay and cell apoptosis assay.Results The expression levels of lncRNA LUC AT1,AR-EG and fibrosis markers α-SMA and COL1A1 were upregulated in endometrial tissues from patients with intrauterine adhesion and in ESC that had been treated with TGF-β1.AREG changed with the change of lncRNA LUCAT1,and the expression of lncRNA LUC-AT1did not change significantly after downregulation of AREG,and AREG was positively regulated by lncRNA LUCAT1.During the process of the transformation of ESC into fibroblasts,si-LUCAT1 significantly inhibited the protein expression levels of AREG,α-SMA and COL1A1(P＜0.01),significantly reduced cell proliferation and significantly induced cell apoptosis,the difference was statistically significant(P＜0.001).pcDNA LUCAT1 significantly induced the protein expression levels of AREG,α-SMA and COL1A1,and the difference was statistically significant(P＜0.01).Conclusion LncRNA LUCAT1 promotes the pathogenesis of intrauterine adhesion by up-regulating the expression of AREG.LncRNA LUCAT1/AREG axis may provide novel molecular target for the prevention and treat-ment of intrauterine adhesion.