Tegoprazan is a novel potassium-competitive acid blocker with long-lasting and potent acid-suppressing effects,showing favorable efficacy in many acid-related diseases.In eradicating Helicobacter pylori(Hp)infections,tegoprazan-based regimens are noninferior to proton pump inhibitor(PPI)regimens in terms of efficacy,and are well tolerated and safe.In this article,we review the pharmacological mechanism,clinical studies on Hp eradication regimens,and safety of tegoprazan to provide a reference for the selection of Hp e-radication regimens.

Objective To screen Coxsackievirus A16(CA16)vaccine candidate strains and provide preliminary basis for the development of vaccine against hand,foot and mouth disease(HFMD).Methods Cell inoculation and continuous passage were performed on the 35 clinical specimens of patients with hand,foot,and mouth disease from different areas of Kunming.The harvested solution with cytopathic effects was identified using CA16 standard serum.Positive samples were screened for vaccine strains through the virus titer determination,plaque purification,VP1 sequence homology and evolutionary analysis.Results Among the 35 collected clinical oropharyngeal swab samples,23 showed cellular lesions,and 10 were identified as CA16 virus by serology,with a positive rate of 43.5%.After the adaptive cultivation,the virus titers of 10 strains gradually increased,but stabilized at 7.0 lgCCID50 from 48 hours onwards.The plaque of the strain showed that all strains can form plaques of 0.2～0.8 cm,showing the regular differences,and the size of KM/M08 plaques was relatively uniform.All strains were B1b type,with nucleotide homology ranging from 93.3～100%.Among them,KM/M08 had the highest homology with other strains,ranging from 95.2～100%.Conclusion Through the analysis of virus proliferation kinetics curve,gene homology,and evolution,KM/M08 meets the expected screening criteria in all aspects of evaluation and is proposed as a vaccine candidate strain for further researches.

Objective To investigate the mechanism by which Yangmai Tongluo formula improves microcirculation disorders induced by high homocysteine(Hcy)levels via regulation of the acid sphingomyelinase(ASM)and endoplasmic reticulum(ER)stress pathways.Methods Fifty male C57BL/6J mice were divided randomly into a control group,Hcy model group,Yangmai Tongluo formula low-/high dose groups(5.3,10.4 g/kg,respectively),and a folic acid group(0.08 g/kg).Except for the control group,microcirculation disorders were induced in all mice using drinking water containing 1.8 g/L Hcy for 6 weeks.After modeling for 2 weeks,mice were administered the corresponding treatments by gavage for 4 weeks.Serum Hey concentrations and the blood perfusion volume of the lower extremity microvessels were measured.Protein expression levels of zonula occludens ZO-1,ZO-2,intercellular adhesion molecule-1(ICAM-1),vascular cell adhesion molecule-1(VCAM-1),ASM,glucose-regulated protein 78(GRP78),and C/EBP homologous protein(CHOP)in the cardiac microvascular endothelium were analyzed using immunofluorescence.Results Serum Hcy levels were significantly increased in the Hcy model group compared with the control group(P＜0.05).Yangmai Tongluo formula did not significantly reduce Hey levels compared with the Hey model group,but blood perfusion in the lower extremities was significantly increased(P＜0.01)and expression levels of ZO-1 and ZO-2 in the cardiac microvascular endothelium were restored(P＜0.001)in the Yangmai Tongluo formula high dose group.It also inhibited the expression of ICAM-1,VCAM-1,ASM,GRP78,and CHOP(P＜0.05),with comparable effects to folic acid.Conclusions Yangmai Tongluo formula improves Hcy-induced microcirculation disorders and endothelial dysfunction by inhibiting ASM activity and alleviating ER stress,via a mechanism closely related to the regulation of endothelial inflammation and barrier stability.These result provide experimental evidence to support the use of traditional Chinese medicine to treat microvascular diseases.

Objective To investigate the mechanism by which Yangmai Tongluo formula improves microcirculation disorders induced by high homocysteine(Hcy)levels via regulation of the acid sphingomyelinase(ASM)and endoplasmic reticulum(ER)stress pathways.Methods Fifty male C57BL/6J mice were divided randomly into a control group,Hcy model group,Yangmai Tongluo formula low-/high dose groups(5.3,10.4 g/kg,respectively),and a folic acid group(0.08 g/kg).Except for the control group,microcirculation disorders were induced in all mice using drinking water containing 1.8 g/L Hcy for 6 weeks.After modeling for 2 weeks,mice were administered the corresponding treatments by gavage for 4 weeks.Serum Hey concentrations and the blood perfusion volume of the lower extremity microvessels were measured.Protein expression levels of zonula occludens ZO-1,ZO-2,intercellular adhesion molecule-1(ICAM-1),vascular cell adhesion molecule-1(VCAM-1),ASM,glucose-regulated protein 78(GRP78),and C/EBP homologous protein(CHOP)in the cardiac microvascular endothelium were analyzed using immunofluorescence.Results Serum Hcy levels were significantly increased in the Hcy model group compared with the control group(P＜0.05).Yangmai Tongluo formula did not significantly reduce Hey levels compared with the Hey model group,but blood perfusion in the lower extremities was significantly increased(P＜0.01)and expression levels of ZO-1 and ZO-2 in the cardiac microvascular endothelium were restored(P＜0.001)in the Yangmai Tongluo formula high dose group.It also inhibited the expression of ICAM-1,VCAM-1,ASM,GRP78,and CHOP(P＜0.05),with comparable effects to folic acid.Conclusions Yangmai Tongluo formula improves Hcy-induced microcirculation disorders and endothelial dysfunction by inhibiting ASM activity and alleviating ER stress,via a mechanism closely related to the regulation of endothelial inflammation and barrier stability.These result provide experimental evidence to support the use of traditional Chinese medicine to treat microvascular diseases.

Objective To explore the mechanism by which puerarin alleviates the cardiotoxicity induced by doxorubicin in myocardial cells. Methods Cells in the logarithmic growth phase were divided into normal control group,model group,low-(20 mmol·L-1),medium-(40 mmol·L-1) and high-(80 mmol·L-1) dose puerarin groups,and positive control group(captopril,1 mmol·L-1). Except for the normal control group,the other groups were co-incubated with 5 mmol·L-1 doxorubicin. Cell viability was assessed using CCK-8 and lactate dehydrogenase (LDH) assays. ROS levels were detected using a ROS probe. Autophagy flux was detected by transfection with HBAD-mcherry-EGFP-LC3 adenovirus. Western Blot was used to measure the protein expression levels of Beclin-1,LC3,p62,p-AMPKα,and AMPKα. Lysosomal function was assessed using a lysosomal probe. Immunofluorescence was used to detect the relative intensity and co-localization of ASMase and LAMP1. Molecular docking analysis was performed to predict the binding capacity of PUE with ASMase. Differential gene expression was analyzed by gene set enrichment analysis. Results Compared to the normal control group,the model group showed reduced cell viability (P<0.01),increased release levels of LDH and ROS (P<0.05,P<0.01),increased number of autophagosomes (P<0.01),and decreased number of autophagic lysosomes (P<0.05). Beclin-1 protein expression and LC3-II/LC3-I ratio decreased(P<0.01),but p62 protein expression increased(P<0.01). Fluorescence intensity of lysosome decreased(P<0.01),whereas fluorescence intensity of ASMase increased(P<0.01). Immunofluorescence co-localization of ASMase and LAMP1 increased (P<0.01),the ratio of p-AMPKα/AMPKα decreased(P<0.05). Compared to the model group,the high-dose puerarin group showed a rebound in cell viability (P<0.05). The medium-and high-dose puerarin groups showed a decreasing trend in LDH level (P<0.05),and all puerarin groups showed a decreasing trend in ROS level (P<0.01). The number of autophagosomes in high-dose puerarin group reduced (P<0.01). The number of autophagic lysosomes in all puerarin groups increased (P<0.05,P<0.01). The high-dose puerarin group showed increased expression of Beclin-1 (P<0.05) and LC3-II/LC3-I ratio,and decreased p62 expression (P<0.01). All puerarin groups showed increased lysosomal fluorescence intensity (P<0.05,P<0.01). The medium-and high-dose puerarin groups showed a decrease in ASMase fluorescence intensity(P<0.05),a reduction in the immunofluorescence co-localization of ASMase with LAMP1 (P<0.01),and an increase in the p-AMPKα/AMPKα ratio (P<0.01). Molecular docking analysis discovered puerarin showed a binding energy of-8.6 kcal·mol-1 with ASMase. Gene enrichment analysis indicated that the differentially expressed genes in the doxorubicin cardiotoxicity model were related to apoptosis,autophagy,and lysosomal function. Conclusion Puerarin can alleviate doxorubicin-induced cardiotoxicity in myocardial cells and protect myocardial cells by regulating autophagy through AMPK/ASMase,as well as restoring autophagic flux.

This study was designed to investigate the effect of silencing microtubule-affinity regulating kinase 4(MARK4)on the apoptosis,inflammatory cytokine release and intestinal barrier protein expression of FHC cells in a lipopolysaccharide(LPS)-induced ulcerative colitis(UC)model,and the underlying molecular mechanisms.Western blot analysis was used to measure the expression levels of MARK4 and apoptosis-related factors including Caspase-1,NLRP3,and GSDMD in colon tissues from both UC patients and healthy individuals,as well as in LPS-induced FHC cell inflammation model.FHC cells was transfected with shRNA to silence MARK4.In control(normal FHC cells),LPS(LPS-stimulated FHC cells),and MARK4-silenced+LPS(shRNA-and LPS-treated FHC cells)groups,the expression levels of Caspase-1,NLRP3,GSDMD,intestinal barrier proteins,and NF-κB pathway-related proteins were assessed by Western blotting.ELISA and RT-qPCR were used to measure the expression levels of inflammatory cytokines IL-1β,IL-6,and TNF-α;flow cytometry was utilized to assess apoptosis.Data showed that both in UC patient colon tissues and the in vitro LPS-induced FHC cell UC inflammation model,there was a significant increase in the expression of MARK4 and apoptosis-related proteins including NLRP3,Caspase-1,and GSDMD.Silencing MARK4 inhibited the expression of these apoptosis-related proteins and downregulated the inflammatory cytokines IL-1β,IL-6,and TNF-α in LPS-induced FHC cells.Silencing MARK4 also reduced apoptosis,increased the expression of intestinal barrier proteins ZO-1,Occludin,and upregulated Claudin2.Gene Set Enrichment Analysis(GSEA)indicated a positive correlation between MARK4 and the NF-κB signaling pathway.Furthermore,silencing of MARK4 inhibited the expression levels of p-P65 and p-IKKα in the NF-κB pathway.In conclusion,MARK4 is significantly upregulated in UC tissues and cells.Silencing MARK4 inhibits the activation of the NF-κB signaling pathway,thereby inhibiting the apoptosis and inflammatory factor expression of UC cells.Thus,MARK4 could be a potential therapeutic target for UC patients.

Lipocalin-2 (LCN2) is a secreted glycoprotein originally purified from mouse kidney cells infected with simian virus 40 and plays a key role in the control of cellular homeostasis during inflammation and the response to cellular stress or injury, and it is considered a potential biomarker for rheumatic diseases, cancer, liver diseases, and inflammatory diseases. Studies have shown that LCN2 is expressed in hepatic parenchymal and nonparenchymal cells and is secreted into the bloodstream, and it is closely associated with the development and progression of acute liver injury, liver cirrhosis, viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease, and hepatocellular carcinoma. This article summarizes the animal experiments and clinical studies on the association of LCN2 with the pathogenesis of liver diseases, in order to provide new ideas and therapeutic targets for the prevention and treatment of liver diseases.

Objective: To observe the effect of differentiated mature adipocytes on hepatic steatosis and aquaporin-9 (AQP9) expressions in HepG2 cells and further explore its possible mechanism of action. Methods: Human preadipocytes were cultured and differentiated to full maturity. HepG2 cells were co-cultured with non-differentiated adipocytes and differentiated mature adipocytes for 48 h, and then labeled as control group and experimental group. Oil red O staining and intracellular triglyceride content were performed on co-cultured HepG2 cells and simultaneous changes in phosphatidylinositol 3-kinase (PI3K) - serine/threonine kinase (Akt) signaling pathway, and AQP9 mRNA and protein levels were detected. The experimental group was co-cultured with recombinant human insulin-like growth factor-I (IGF-I), with the addition of 100ng/ml PI3K-Akt pathway agonist, labeled as experimental group + IGF-I group. The activation of PI3K-Akt pathway was verified by Western blotting (WB). The expression of AQP9 was detected by RT-q PCR and WB. The recombinant lentivirus LV-AQP9 or empty-loaded virus LV-PWPI was transfected with HepG2 cells by recombinant lentiviral transfection tecnique, and labeled as HepG2-AQP9 and HepG2-PWPI. The transfection efficiency was assessed by confocal laser scanning microscopy and RT-qPCR and WB detected the change of AQP9 expression level after virus transfection. Afterwards, the stable over-expressed HepG2-AQP9 cells and the empty-loaded HepG2-PWPI cells were co-cultured with differentiated mature adipocytes for 48h, and labeled as HepG2-AQP9 co-culture group, and then intracellular triglyceride content were detected with Oil red O staining. Finally, IGF-I was added to the HepG2-AQP9 co-culture group, which was recorded as HepG2-AQP9 co-culture + IGF-I group. Intracellular triglyceride content was detected with Oil red O staining, and WB verified PI3K-Akt signaling pathway activation and changes in AQP9 mRNA and protein levels. A t-test was used to compare the two independent samples. Results: The intracellular lipid droplets and triglyceride content (0.052 ± 0.005) in the experimental group was increased significantly than the control group (0.033 ± 0.003) (t= 5.225,P= 0.006), suggesting that adipocyte co-culture had induced steatosis in HepG2 cells. RT-qPCR and WB results indicated that the expression levels of AQP9 mRNA (3.615 ± 0.330) and protein levels (0.072 ± 0.005) in the experimental group were significantly higher than the control group (t= 13.708, 11.225,P= 0.005, < 0.001). WB results showed that the expression level of phosphorylated Akt (p-Akt) protein (0.116±0.003) in the experimental group was significantly lower than the control group (0.202 ± 0.003) (t= 27.136,P< 0.001). The total Akt protein was constant, and the p-Akt/total Akt (0.182 ± 0.017)was significantly lower than the control group (0.327 ± 0.019) (t= 2.431,P= 0.001), suggesting that adipocyte co-culture had inhibited PI3K- Akt signaling pathway in HepG2 cells and up-regulated the expression level of AQP9. WB results indicated that the expression level of p-Akt protein (0.194 ± 0.021) in the experimental group + IGF-I group was significantly higher than the experimental group (0.132 ± 0.003) (t= 5.082,P= 0.007). The total Akt protein was constant, and the p-Akt/total Akt (0.281 ± 0.009) was significantly higher than the control group (0.184 ± 0.132) (t= 10.311,P< 0.001). Simultaneously, RT-qPCR and WB results indicated that the expression levels of AQP9 mRNA (0.327 ± 0.347) and protein levels (0.042 ± 0.004) in the experimental group + IGF-I group were significantly lower than the experimental group (t= 33.573, 5.598,P< 0.001, 0.005), suggesting that adipocyte co-culture had possibility to regulate the expression level of AQP9 through the PI3K-Akt pathway. Confocal laser microscopy analysis showed that the transfection efficiency was more than 90%. RT-q PCR and WB results indicated that the expression levels of AQP9 mRNA and protein levels (0.373 ± 0.221) in HepG2-AQP9 group were significantly higher than HepG2-PWPI group (t=14.953, 28.931,P= 0.002 and 0.000), suggesting that the stable overexpression of AQP9 cell line was successfully constructed. The intracellular lipid droplets and triglyceride content in HepG2-AQP9 co-culture group was significantly increased (t= 5.478, 5.369,P= 0.005) than HepG2-PWPI co-culture group and HepG2-AQP9 co-culture+ IGF-I group, suggesting that the increased expression of AQP9 had promoted HepG2 steatosis in co-cultured adipocytes. WB results showed the expression levels of p-Akt protein (0.168 ± 0.006) and p-Akt/total Akt (0.265±0.009) in HepG2-AQP9 co-culture + IGF-1 group was significantly increased (t= 16.311, 8.769,P< 0.001) than HepG2-AQP9 co-culture group, while the expression levels of AQP9 mRNA (0.327 ± 0.034) and protein (0.375 ± 0.025) was significantly decreased (t= 33.573, 9.146,P< 0.001 and 0.001). Conclusion Adipocytes co-culture can induce steatosis in HepG2 cells, and may participate in inhibiting PI3K-Akt signaling pathway to upregulate the expression of AQP9 in steatotic HepG2 cells.

Objective To observe and analyze the pathological changes in BALB/c mice infected with herpes simplex virus typeⅡ (HSV-2) through nasal and genital inoculation. Methods Six-week old female BALB/c mice were divided into two groups, experimental and control groups. In the experimental group, the mice were infected with HSV-2 (104 CCID50/20μl per mouse) through nasal and genital tract in-oculation. Accordingly, the mice in the control group were injected with equal volume of PBS. Tissue speci-mens were collected from lung, nervous system and reproductive system for pathological analysis and viral load detection at different time points after infection. Lat gene expression in mouse trigeminal and sacral gan-glia was detected through in situ hybridization. In addition, the proliferation of viruses isolated form trigemi-nal and sacral ganglia of the infected mice was observed in vitro. Results Weight loss and histopathological lesions were observed in the mice of the experimental group 6 d after infection. Major pathological changes in the HSV-2-infected mice through nasal tract inoculation involved the lung and central nervous system( CNS) , including alveolar wall congestion, cerebrovascular cuff response and lymphocyte infiltration. How-ever, the major lesions in the infected mice through genital tract inoculation were found in the reproductive ducts, such as sacral ganglion necrosis, eosinophilia in the vagina and uterus, and ovarian congestion. Re-sults of the viral load detection in tissues and organs of the infected mice were consistent with the pathological changes. The mice infected through nasal tract inoculation had significantly higher viral loads in the nerves and lungs than those by genital tract inoculation, but lower viral loads in the genital tracts and sacral ganglia. Positive expression of lat gene at mRNA level was detected in the trigeminal and sacral ganglia of mice with HSV-2 latency 28 d after infection. In addition, both of the tissue fragments from trigeminal and sacral ganglia had cytopathic effects ( CPEs) on Vero cells. Enhanced expression of lat gene at mRNA level and much severer CPEs were induced by genital tract inoculation than by nasal tract inoculation. Conclu-sions HSV-2 could infect and cause histopathological damages in BALB/c mice through both nasal and genital tracts. In addition, the locations of the pathological lesions were closely related to the mode of infection.

Objective To investigate the influences of herpes simplex virus 1 and 2 ( HSV1 and HSV2) infection on the expression of signaling molecules associated with innate immune response in respira-tory and vaginal epithelial cells for bettering understanding of HSV infection and pathological characteristics in the primary infection site, namely mucosal epithelial tissues. Methods KMB17 and VK2 cells were in-fected with HSV. Changes in cell morphology and inner structure after HSV infection were observed under optical microscope and scanning electron microscope, respectively. Viral proliferation in KMB17 and VK2 cells was detected by plaque assay, microcytopathic assay and real-time quantitative PCR. Expression of sig-naling molecules associated with innate immune response in virus-infected KMB17 and VK2 cells were ana-lyzed by real-time quantitative PCR. Results Both HSV1 and HSV2 could infect KMB17 and VK2 cells, and cause damage to cell morphology and inner structure after 12 hours. Both of the two viruses formed simi-lar plaque on the single layer of KMB17 and VK2 cells, although HSV2 proliferated slower than HSV1. There were differences in the expression of signaling molecules associated with innate immune response in-duced by the two viruses in KMB17 and VK2 cells. Conclusion Both HSV1 and HSV2 could infect and proliferate in epithelial cells ( KMB17 and VK2 cells) . Although there were slight differences in viral prolif-eration between them, significant differences in the expression of signaling molecules associated with innate immune response induced by the two viruses were observed.