BACKGROUND:Formononetin demonstrates potent anti-inflammatory and antioxidant abilities.However,its protective effect on nucleus pulposus mesenchymal stem cells is not yet clear.OBJECTIVE:To investigate the effect and mechanism of formononetin on nucleus pulposus mesenchymal stem cells under an inflammatory microenvironment.METHODS:(1)Primary nucleus pulposus mesenchymal stem cells were isolated from the intervertebral discs of SD rats,and flow cytometry was performed to identify the surface markers of mesenchymal stem cells.(2)The CCK-8 assay was employed to evaluate the impact of lipopolysaccharide and formononetin on the proliferation viability of nucleus pulposus mesenchymal stem cells,aiming to determine the appropriate concentration of formononetin for subsequent cell treatments.(3)An inflammatory microenvironment was simulated by adding 5 μg/mL lipopolysaccharide to the DMEM/F-12 culture medium,and nucleus pulposus mesenchymal stem cells were treated with different concentrations of formononetin for 24 hours.Levels of inflammation markers were detected using western blot assay,real-time quantitative PCR,and immunofluorescence.Western blot assay was conducted to measure the protein levels of the nuclear factor kappa B signaling pathway.RESULTS AND CONCLUSION:(1)The nucleus pulposus mesenchymal stem cells cultured in adherent wall were shuttle-shaped with good growth status.The results of flow cytometry showed that the surface markers of mesenchymal stem cells were positive for CD29,CD44,and CD90,and the surface markers of hematopoietic stem cells were negative for CD34 and CD45.(2)The treatment with formononetin at 12.5,25,50,100,and 200 μmol/L concentrations for 24 hours had no significant proliferation inhibitory effect on nucleus pulposus mesenchymal stem cells.Compared with the lipopolysaccharide group,the cell viability of nucleus pulposus mesenchymal stem cells treated with 12.5,25,and 50 μmol/L formononetin for 24 hours was significantly increased,so formononetin at 12.5,25,and 50 μmol/L concentrations was subsequently selected as the low,medium,and high concentrations for treating nucleus pulposus mesenchymal stem cells.(3)Compared with the lipopolysaccharide group,the protein and mRNA expressions of matrix metalloproteinase-3,matrix metalloproteinase-13,and tumor necrosis factor-α in nucleus pulposus mesenchymal stem cells in the low,medium,and high concentrations of formononetin groups were significantly decreased(P＜0.05)in a dose-dependent manner.(4)Compared with the lipopolysaccharide group,the expressions of phosphorylated nuclear factor kappa B and phosphorylated nuclear factor kappa B inhibitor protein in nucleus pulposus mesenchymal stem cells in the low,medium,and high concentrations of formononetin groups were significantly decreased(P＜0.05)in a dose-dependent manner.The above results suggest that formononetin may attenuate lipopolysaccharide-induced inflammation in rat nucleus pulposus mesenchymal stem cells by inhibiting the activation of the nuclear factor kappa B signaling pathway.

AIM To investigate the protective effects of kaempferide on hyperuricemic nephropathy(HN)in mice.METHODS Sixty Kunming mice were randomly divided into the control group,the model group,the allopurinol group(5 mg/kg),the kaempferol group(50 mg/kg),and the low-dose and high-dose kaempferide groups(25,50 mg/kg).HN mouse models were established by administering potassium oxyzinate(300 mg/kg)and hypoxanthine(500 mg/kg)in combination for 21 days,concurrently with the test drug.Following treatment administration,serum uric acid(SUA),serum creatinine(SCr),24-hour urinary protein(24 h UTP),and hepatic xanthine oxidase(XOD)levels were measured.Renal tissue pathology was assessed using HE staining and Masson staining.Apoptosis in renal tissue was evaluated via TUNEL staining.The expressions of NLRP3 inflammasome and apoptosis-associated proteins in renal tissues were analyzed by immunohistochemistry and Western blot.RESULTS Compared to the control group,the model group demonstrated elevated levels of SUA,SCr,24 h UTP,and hepatic XOD activity(P＜0.01);marked renal damage,and increased area of renal interstitial fibrosis and apoptosis rate(P＜0.01);and increased protein expressions of NLRP3,ASC,Caspase-1,cleaved-Caspase-1,pro-IL-1β,IL-1β,Caspase-3 and cleaved-Caspase-3 in renal tissue(P＜0.05,P＜0.01).Compared to the model group,the groups treated with allopurinol,kaempferol,or kaempferid showed reduced levels of SUA,SCr,24 h UTP,and hepatic XOD activity(P＜0.05,P＜0.01);improved renal pathological injury with reduced renal interstitial fibrosis area and apoptosis rate of renal tissue(P＜0.01);and downregulated protein expressions ofNLRP3,ASC,Caspase-1,cleaved-Caspase-1,pro-IL-1β,IL-1β,Caspase-3 and cleaved-Caspase-3 in renal tissue as well(P＜0.05,P＜0.01).CONCLUSION Kaempferide improves renal function while attenuating inflammation,fibrosis,and apoptosis in the kidneys of HN mice.This nephroprotective effect may stem from its dual action in inhibiting hepatic XOD to reduce uric acid synthesis and blocking NLRP3 inflammasome activation.

BACKGROUND:Studies have shown that upregulation of heme oxygenase-1 expression enhances cellular antioxidant and anti-apoptotic abilities.However,the effects of upregulating heme oxygenase-1 expression on the proliferation and differentiation of neural stem cells under oxidative stress conditions remain unclear.OBJECTIVE:To investigate the influence of heme oxygenase-1 overexpression on the survival and differentiation capacity of neural stem cells under oxidative stress conditions.METHODS:(1)Mouse primary neural stem cells were isolated and cultured from newborn Balb/c mice.Immunofluorescence was used to detect the neural stem cell marker Nestin.(2)Lentivirus was used to infect neural stem cells to induce heme oxygenase-1 overexpression.Flow cytometry was used to assess green fluorescent protein fluorescence.Western blot assay was performed to detect the expression levels of heme oxygenase-1.(3)H2O2 was added to the lentivirus-infected neural stem cell culture medium to simulate the oxidative stress microenvironment after spinal cord injury.Effects of heme oxygenase-1 overexpression on neural stem cell proliferation and apoptosis levels were analyzed using cell proliferation assay kits,cell apoptosis assay kits,and TUNEL staining kits.(4)The levels of lipid oxidation markers malondialdehyde,catalase,superoxide dismutase,and glutathione peroxidase were detected using assay kits.(5)Flow cytometry was used to measure intracellular reactive oxygen species levels,and neural stem cell differentiation into astrocytes and neurons.(6)The effect of heme oxygenase-1 overexpression on neuronal axon growth during neural stem cell differentiation was observed under optical and fluorescence microscopes.RESULTS AND CONCLUSION:(1)Mouse neural stem cells exhibited stable morphology,good growth status,and high expression of Nestin as detected by immunofluorescence.(2)Western blot analysis showed that the overexpression of heme oxygenase-1 in the overexpression group was significantly higher than that in the empty carrier control group.Flow cytometry was used to detect the expression of green fluorescent protein in the neural stem cells of the heme oxygenase-1 overexpression group and empty vector control group.(3)Overexpression of heme oxygenase-1 maintained the proliferative activity of neural stem cells and significantly reduced the number of apoptotic cells under oxidative stress conditions.(4)Overexpression of heme oxygenase-1 inhibited lipid peroxidation of neural stem cells under oxidative stress microenvironment,enhanced the expression of enzymes related to maintaining the oxidative-reductive balance,and significantly reduced intracellular reactive oxygen species levels.(5)Overexpression of heme oxygenase-1 promoted the differentiation of neural stem cells into neurons and reduced differentiation into astrocytes.(6)The heme oxygenase-1 overexpression group exhibited longer axons,and more intercellular connections.The above results indicate that overexpression of heme oxygenase-1 can alleviate oxidative damage of H2O2-induced neural stem cells,reduce neural stem cell apoptosis,promote proliferation,and facilitate differentiation of neural stem cells into neurons.

AIM To investigate the protective effects of kaempferide on hyperuricemic nephropathy(HN)in mice.METHODS Sixty Kunming mice were randomly divided into the control group,the model group,the allopurinol group(5 mg/kg),the kaempferol group(50 mg/kg),and the low-dose and high-dose kaempferide groups(25,50 mg/kg).HN mouse models were established by administering potassium oxyzinate(300 mg/kg)and hypoxanthine(500 mg/kg)in combination for 21 days,concurrently with the test drug.Following treatment administration,serum uric acid(SUA),serum creatinine(SCr),24-hour urinary protein(24 h UTP),and hepatic xanthine oxidase(XOD)levels were measured.Renal tissue pathology was assessed using HE staining and Masson staining.Apoptosis in renal tissue was evaluated via TUNEL staining.The expressions of NLRP3 inflammasome and apoptosis-associated proteins in renal tissues were analyzed by immunohistochemistry and Western blot.RESULTS Compared to the control group,the model group demonstrated elevated levels of SUA,SCr,24 h UTP,and hepatic XOD activity(P＜0.01);marked renal damage,and increased area of renal interstitial fibrosis and apoptosis rate(P＜0.01);and increased protein expressions of NLRP3,ASC,Caspase-1,cleaved-Caspase-1,pro-IL-1β,IL-1β,Caspase-3 and cleaved-Caspase-3 in renal tissue(P＜0.05,P＜0.01).Compared to the model group,the groups treated with allopurinol,kaempferol,or kaempferid showed reduced levels of SUA,SCr,24 h UTP,and hepatic XOD activity(P＜0.05,P＜0.01);improved renal pathological injury with reduced renal interstitial fibrosis area and apoptosis rate of renal tissue(P＜0.01);and downregulated protein expressions ofNLRP3,ASC,Caspase-1,cleaved-Caspase-1,pro-IL-1β,IL-1β,Caspase-3 and cleaved-Caspase-3 in renal tissue as well(P＜0.05,P＜0.01).CONCLUSION Kaempferide improves renal function while attenuating inflammation,fibrosis,and apoptosis in the kidneys of HN mice.This nephroprotective effect may stem from its dual action in inhibiting hepatic XOD to reduce uric acid synthesis and blocking NLRP3 inflammasome activation.

BACKGROUND:Formononetin demonstrates potent anti-inflammatory and antioxidant abilities.However,its protective effect on nucleus pulposus mesenchymal stem cells is not yet clear.OBJECTIVE:To investigate the effect and mechanism of formononetin on nucleus pulposus mesenchymal stem cells under an inflammatory microenvironment.METHODS:(1)Primary nucleus pulposus mesenchymal stem cells were isolated from the intervertebral discs of SD rats,and flow cytometry was performed to identify the surface markers of mesenchymal stem cells.(2)The CCK-8 assay was employed to evaluate the impact of lipopolysaccharide and formononetin on the proliferation viability of nucleus pulposus mesenchymal stem cells,aiming to determine the appropriate concentration of formononetin for subsequent cell treatments.(3)An inflammatory microenvironment was simulated by adding 5 μg/mL lipopolysaccharide to the DMEM/F-12 culture medium,and nucleus pulposus mesenchymal stem cells were treated with different concentrations of formononetin for 24 hours.Levels of inflammation markers were detected using western blot assay,real-time quantitative PCR,and immunofluorescence.Western blot assay was conducted to measure the protein levels of the nuclear factor kappa B signaling pathway.RESULTS AND CONCLUSION:(1)The nucleus pulposus mesenchymal stem cells cultured in adherent wall were shuttle-shaped with good growth status.The results of flow cytometry showed that the surface markers of mesenchymal stem cells were positive for CD29,CD44,and CD90,and the surface markers of hematopoietic stem cells were negative for CD34 and CD45.(2)The treatment with formononetin at 12.5,25,50,100,and 200 μmol/L concentrations for 24 hours had no significant proliferation inhibitory effect on nucleus pulposus mesenchymal stem cells.Compared with the lipopolysaccharide group,the cell viability of nucleus pulposus mesenchymal stem cells treated with 12.5,25,and 50 μmol/L formononetin for 24 hours was significantly increased,so formononetin at 12.5,25,and 50 μmol/L concentrations was subsequently selected as the low,medium,and high concentrations for treating nucleus pulposus mesenchymal stem cells.(3)Compared with the lipopolysaccharide group,the protein and mRNA expressions of matrix metalloproteinase-3,matrix metalloproteinase-13,and tumor necrosis factor-α in nucleus pulposus mesenchymal stem cells in the low,medium,and high concentrations of formononetin groups were significantly decreased(P＜0.05)in a dose-dependent manner.(4)Compared with the lipopolysaccharide group,the expressions of phosphorylated nuclear factor kappa B and phosphorylated nuclear factor kappa B inhibitor protein in nucleus pulposus mesenchymal stem cells in the low,medium,and high concentrations of formononetin groups were significantly decreased(P＜0.05)in a dose-dependent manner.The above results suggest that formononetin may attenuate lipopolysaccharide-induced inflammation in rat nucleus pulposus mesenchymal stem cells by inhibiting the activation of the nuclear factor kappa B signaling pathway.

BACKGROUND:Studies have shown that upregulation of heme oxygenase-1 expression enhances cellular antioxidant and anti-apoptotic abilities.However,the effects of upregulating heme oxygenase-1 expression on the proliferation and differentiation of neural stem cells under oxidative stress conditions remain unclear.OBJECTIVE:To investigate the influence of heme oxygenase-1 overexpression on the survival and differentiation capacity of neural stem cells under oxidative stress conditions.METHODS:(1)Mouse primary neural stem cells were isolated and cultured from newborn Balb/c mice.Immunofluorescence was used to detect the neural stem cell marker Nestin.(2)Lentivirus was used to infect neural stem cells to induce heme oxygenase-1 overexpression.Flow cytometry was used to assess green fluorescent protein fluorescence.Western blot assay was performed to detect the expression levels of heme oxygenase-1.(3)H2O2 was added to the lentivirus-infected neural stem cell culture medium to simulate the oxidative stress microenvironment after spinal cord injury.Effects of heme oxygenase-1 overexpression on neural stem cell proliferation and apoptosis levels were analyzed using cell proliferation assay kits,cell apoptosis assay kits,and TUNEL staining kits.(4)The levels of lipid oxidation markers malondialdehyde,catalase,superoxide dismutase,and glutathione peroxidase were detected using assay kits.(5)Flow cytometry was used to measure intracellular reactive oxygen species levels,and neural stem cell differentiation into astrocytes and neurons.(6)The effect of heme oxygenase-1 overexpression on neuronal axon growth during neural stem cell differentiation was observed under optical and fluorescence microscopes.RESULTS AND CONCLUSION:(1)Mouse neural stem cells exhibited stable morphology,good growth status,and high expression of Nestin as detected by immunofluorescence.(2)Western blot analysis showed that the overexpression of heme oxygenase-1 in the overexpression group was significantly higher than that in the empty carrier control group.Flow cytometry was used to detect the expression of green fluorescent protein in the neural stem cells of the heme oxygenase-1 overexpression group and empty vector control group.(3)Overexpression of heme oxygenase-1 maintained the proliferative activity of neural stem cells and significantly reduced the number of apoptotic cells under oxidative stress conditions.(4)Overexpression of heme oxygenase-1 inhibited lipid peroxidation of neural stem cells under oxidative stress microenvironment,enhanced the expression of enzymes related to maintaining the oxidative-reductive balance,and significantly reduced intracellular reactive oxygen species levels.(5)Overexpression of heme oxygenase-1 promoted the differentiation of neural stem cells into neurons and reduced differentiation into astrocytes.(6)The heme oxygenase-1 overexpression group exhibited longer axons,and more intercellular connections.The above results indicate that overexpression of heme oxygenase-1 can alleviate oxidative damage of H2O2-induced neural stem cells,reduce neural stem cell apoptosis,promote proliferation,and facilitate differentiation of neural stem cells into neurons.

Objective To investigate the clinical efficacy of Guiyuan Shujin Mixture in the treatment of patients with lumbar disc herniation(LDH)and to explore its possible therapeutic mechanism.Methods Sixty-eight patients with LDH of qi stagnation and blood stasis syndrome were randomly divided into trial group and control group,with 34 cases in each group.The control group was treated with Celecoxib Tablets and Mecobalamin Tablets orally,and the trial group was treated with Guiyuan Shujin Mixture on the basis of treatment for the control group.The course of treatment lasted for 4 weeks.Before and after treatment,the two groups were observed in the changes of the Visual Analogue Scale(VAS)score of low back pain and lower limb pain,Oswestry Disability Index(ODI)score,modified Japanese Orthopedic Association(JOA)score,serum levels of inflammatory factors of tumor necrosis factor α(TNF-α),interleukin 6(IL-6)and interleukin 1β(IL-1β),and serum nuclear factor-κB p65(NF-κB p65)level.After treatment,the clinical efficacy and safety of the two groups were evaluated.Results(1)During the trial,one case fell off in the trial group and 3 cases fell off in the control group.Eventually,33 cases in the trial group and 31 cases in the control group were included for the efficacy statistics.(2)After 4 weeks of treatment,the total effective rate of the trial group was 96.97%(32/33),and that of the control group was 87.10%(27/31).The intergroup comparison(tested by rank sum test)showed that the curative effect of the trial group was significantly superior to that of the control group(P＜0.05).(3)After treatment,the VAS score and ODI score of low back pain and lower limb pain in the two groups were lower than those before treatment(P＜0.05 or P＜0.01),and the modified JOA score was higher than that before treatment(P＜0.01).The decrease of VAS score and ODI score of low back pain and lower limb pain and the increase of modified JOA score in the trial group were significantly superior to those in the control group(P＜0.05 or P＜0.01).(4)After treatment,the serum levels of inflammation-related indicators of TNF-α,IL-6,IL-1β and NF-κB p65 in the two groups were lower than those before treatment(P＜0.01),and the decrease in the trial group was significantly superior to that in the control group(P＜0.01).(5)During the treatment,the incidence of adverse events in the trial group was 2.94%(1/34)and that in the control group was 8.82%(3/34),and the difference between the two groups was not significant(P＞0.05).Conclusion Guiyuan Shujin Mixture exerts certain effect in the treatment of LDH patients with qi stagnation and blood stasis syndrome.It can effectively relieve the pain symptoms of patients,improve the lumbar function of patients,and reduce the expression levels of serum inflammatory factors and NF-κB p65.The mechanism may be related with the decrease of the level of inflammatory factors and with the inhibition of the activation of NF-κB signaling pathway.

Objective:To explore the effect of early clinical clerkship training in the early exposure to clinical practice of eight-year program medical students.Methods:Experimental control method was used in this study. A total of 120 eight-year program medical students in the third year of Batch 2014 to 2016 from the Union Hospital, Tongji Medical College, Huazhong University of Science and Technology were selected as the research objects. The students of each grade were randomly selected by computer and divided into experimental group and control group, with 20 students in each group. Before early clinical exposure, the experimental group received 16 class hours of early clinical clerkship training, while the control group did not receive early clinical clerkship training. After the early clinical exposure, the clinical diagnosis and treatment ability of the two groups of students was compared. SPSS 24.0 software was used for t test. Results:The scores of medical history inquiry of experimental group and control group were [(17.45±1.96) points and (15.95±1.93) points; (18.30±1.03) points and (16.75±1.86) points; (17.95±1.36) points and (16.40±1.60) points, respectively]. The physical examination scores were [(17.75±1.65) points and (16.05±1.64) points; (17.85±1.18) points and (16.80±1.47) points; (18.25±1.16) points and (16.85±1.63) points, respectively]. The clinical judgment scores were [(18.15±1.42) points and (16.35±2.41) points; (18.20±1.24) points and (16.65±1.53) points; (18.35±1.35) points and (16.25±1.83) points, respectively]. Diagnosis and treatment scheme scores were [(17.15±1.57) points and (14.55±2.56) points; (17.30±1.42) points and (15.90±1.48) points; (17.80±1.06) points and (16.35±1.87) points, respectively]. The scores of communication skills were [(17.95±1.15) points and (17.00±1.19) points; (18.55±0.83) points and (17.45±1.50) points; (18.45±1.00) points and (17.45±1.23) points, respectively], with statistically significant differences (all P<0.05). Conclusion:The application of early clinical clerkship training in the early exposure to clinical practice of eight-year program medical students can improve the quality of students' clerkship.

OBJECTIVETo establish a mouse model for human chronic HBV infection, and to investigate the role of PD-1/PD-L1 signaling pathway in antiviral immunity.

METHODSA mouse model was established by hydrodynamic injection of the plasmid pAAV/HBV1.2-GFP into the tail vein of C57BL/6 mice, HBV markers were assayed at different time points after injection. After intraperitoneal injection of anti-PD-L1 monoclonal antibody, the serum ALT, and HBV DNA in the serum, liver and kidney were assayed.

RESULTSThe chronic HBV infection mouse model were established successfully, serum HBsAg and high load of HBV DNA were detectable 90 days after plasmid injection. After blocking of the PD-1/PD-L1 pathway, the serum ALT level of mice were significantly increased (P < 0.01), and the HBV DNA load in serum (P < 0.01), liver (P < 0.05) and kidney (P < 0.05) were decreased significantly.

CONCLUSIONBlocking the PD-1/PD-L1 signaling pathway can enhance antiviral response in mice with chronic HBV infection.

Animals ; Antigens, Surface ; metabolism ; Apoptosis Regulatory Proteins ; metabolism ; B7-1 Antigen ; metabolism ; B7-H1 Antigen ; DNA, Viral ; analysis ; Disease Models, Animal ; Hepatitis B virus ; metabolism ; Hepatitis B, Chronic ; immunology ; metabolism ; virology ; Male ; Membrane Glycoproteins ; metabolism ; Mice ; Mice, Inbred C57BL ; Peptides ; metabolism ; Programmed Cell Death 1 Receptor ; Signal Transduction

The anti-hepatitis B virus (HBV) effects and its mechanisms of the ethanol extracts of Hypericum perforatum L. (EHP) in vitro were explored. HepG2 2.2.15 cells, a stable HBV-producing cell line, were cultured as the model system to observe the anti-HBV effect. The viral antigens of cellular secretion, HBsAg and HBeAg, were determined by enzyme linked immunosorbent assay (ELISA). The quantity of HBV-DNA released in the supernatant was assayed by real-time PCR. In order to understand the mechanisms of the suppression of HBV replication, all HBV promoters (Cp, Xp, S1p, S2p and Fp) with luciferase reporter gene were transfected into HepG2 cells respectively. Then the activities of viral promoters were examined by luciferase reporter assay. It was found EHP effectively suppressed the secretion of HBsAg and HBeAg from HepG2 2.2.15 cells in a dose-dependent manner, as well as the extracellular HBV DNA. And EHP could selectively inhibit the activity of HBV promoter Fp. Our data suggest that EHP exerts anti-HBV effects via inhibition of HBV transcription, which helps to elucidate the mechanism underlying the potential therapeutic value of EHP.