OBJECTIVE To investigate the effect of betulin (BE) on the replication of rotavirus (RV) in vitro.METHODS ①MA104 cells were intervened with BE between 0.03125 and 64μmol·L-1 for 72 h,and MTT assay was used to detect the cell survival.② MA104 cells were infected with RV including Wa strains and SA-11 strains to establish a viral model.MA104 cells were divided into the cell control group,RV group and RV+BE groups.The cytopathic effect (CPE) method combined with MTT assay was used to detect the effect of BE on anti-RV adsorption,anti-RV biosynthesis and direct inhibition of RV.③The grouping was the same as in②,and RT-qPCR,Western blot and IF methods were used to detect the expression of RV structural protein VP6 after 24 h of BE incubation.④The grouping was the same as in②,and the ELISA kit was used to detect the concentrations of pro-inflammatory factors IL-6,IL-1β and TNF-α,anti-inflammatory factor IL-10 and type Ⅱ interferon IFN-γ in the cell supernatant after 24 h of BE incubation.⑤The grouping was the same as in②,and qPCR assay was used to detect the mRNA expression levels of Toll-like receptor 4 (TLR4),myeloid differentiation factor 88 (MYD88) and TNF receptor-associated factor 6 (TRAF6) after 24 h of BE incubation,Western blot assay was used to detect the expression levels of MYD88,IκBα,NF-κB p65,and p-NF-κB p65 after 24 h of BE incubation.RESULTS ① The maximum non-toxic concentration of BE towards MA104 cells was 1 μmol·L-1,and TC50 was 5.795 μmol·L-1.The concentrations of BE in the anti-RV experiments ranged from 0.03125μmol·L-1 to 1μmol·L-1.② In the anti-RV biosynthesis experiment,the inhibition rates of BE for RV-Wa between 0.0625 and 1 μmol·L-1 exceeded 50％,EC50 was 0.0485 μmol·L-1,and the value of TI was 119.48.The inhibition rates of BE for RV-SA-11 between 0.25 and 1 μmol·L-1 were above 50％,EC50 was 0.1226 μmol·L-1,and the value of TI was 47.27.In contrast,the effects of BE on anti-RV adsorption and direct inhibition of RV were not obvious.③ Compared with the RV group,BE inhibited the expression of VP6 (P＜0.01).④ Compared with the RV group,BE reduced the concentrations of IL-6,IL-1β and TNF-α(P＜0.01),but increased the concentrations of IL-10 and IFN-γ(P＜0.01).⑤Compared with the RV group,BE reduced the mRNA levels of TLR4,MYD88 and TRAF6 (P＜0.01),decreased the protein expression levels of MYD88 and p-NF-κB p65,and increased those of IκBα and NF-κB p65 (P＜0.01).CONCLUSION BE has anti-RV biosynthesis effect,and it may reduce inflammatory response caused by RV infection via TLR4/MYD88/NF-κB signaling pathway.

OBJECTIVE To investigate the effect of betulin (BE) on the replication of rotavirus (RV) in vitro.METHODS ①MA104 cells were intervened with BE between 0.03125 and 64μmol·L-1 for 72 h,and MTT assay was used to detect the cell survival.② MA104 cells were infected with RV including Wa strains and SA-11 strains to establish a viral model.MA104 cells were divided into the cell control group,RV group and RV+BE groups.The cytopathic effect (CPE) method combined with MTT assay was used to detect the effect of BE on anti-RV adsorption,anti-RV biosynthesis and direct inhibition of RV.③The grouping was the same as in②,and RT-qPCR,Western blot and IF methods were used to detect the expression of RV structural protein VP6 after 24 h of BE incubation.④The grouping was the same as in②,and the ELISA kit was used to detect the concentrations of pro-inflammatory factors IL-6,IL-1β and TNF-α,anti-inflammatory factor IL-10 and type Ⅱ interferon IFN-γ in the cell supernatant after 24 h of BE incubation.⑤The grouping was the same as in②,and qPCR assay was used to detect the mRNA expression levels of Toll-like receptor 4 (TLR4),myeloid differentiation factor 88 (MYD88) and TNF receptor-associated factor 6 (TRAF6) after 24 h of BE incubation,Western blot assay was used to detect the expression levels of MYD88,IκBα,NF-κB p65,and p-NF-κB p65 after 24 h of BE incubation.RESULTS ① The maximum non-toxic concentration of BE towards MA104 cells was 1 μmol·L-1,and TC50 was 5.795 μmol·L-1.The concentrations of BE in the anti-RV experiments ranged from 0.03125μmol·L-1 to 1μmol·L-1.② In the anti-RV biosynthesis experiment,the inhibition rates of BE for RV-Wa between 0.0625 and 1 μmol·L-1 exceeded 50％,EC50 was 0.0485 μmol·L-1,and the value of TI was 119.48.The inhibition rates of BE for RV-SA-11 between 0.25 and 1 μmol·L-1 were above 50％,EC50 was 0.1226 μmol·L-1,and the value of TI was 47.27.In contrast,the effects of BE on anti-RV adsorption and direct inhibition of RV were not obvious.③ Compared with the RV group,BE inhibited the expression of VP6 (P＜0.01).④ Compared with the RV group,BE reduced the concentrations of IL-6,IL-1β and TNF-α(P＜0.01),but increased the concentrations of IL-10 and IFN-γ(P＜0.01).⑤Compared with the RV group,BE reduced the mRNA levels of TLR4,MYD88 and TRAF6 (P＜0.01),decreased the protein expression levels of MYD88 and p-NF-κB p65,and increased those of IκBα and NF-κB p65 (P＜0.01).CONCLUSION BE has anti-RV biosynthesis effect,and it may reduce inflammatory response caused by RV infection via TLR4/MYD88/NF-κB signaling pathway.

Objective:To evaluate the efficacy and safety of transhepatic arterial chemoembolization (TACE) combined with tyrosine kinase inhibitors (TKI) and programmed death-1 (PD-1) inhibitors in the treatment of patients with initially unresectable hepatocellular carcinoma.Methods:The clinical data of 42 patients with initially unresectable hepatocellular carcinoma who were admitted to the Department of Hepatobiliary and Pancreatic Surgery of the First Affiliated Hospital of Zhengzhou University from January 2020 to December 2022 were included. There were 31 males and 11 females, with a median age of 56 years old (range, 45-72 years old). All patients received TACE+ TKI+ PD-1 inhibitor combined treatment. The systemic treatment cycles were calculated by the regimen of immunotherapy. The timing of local treatment depends on tumor size, blood supply and treatment response. Patients were followed up through hospitalization, outpatient visits and telephone review. The Kaplan-Meier curves were obtained for survival analysis.Results:The dosing cycle to achieve optimal imaging response in the patients was 4 (3, 7) [ M( Q1, Q3)], with a systemic treatment time of 141 (65, 194) d [ M( Q1, Q3)] and 2 (1, 3) times [ M( Q1, Q3)] of local treatments. All patients were evaluated by modified response evaluation criteria in solid tumors criteria after treatment, including nine patients with complete response (CR), 21 with partial response, eight with stable disease, and four with progressive disease. Objective response rate and disease control rate were 71.4% (30/42) and 90.5% (38/42), respectively. Treatment-related adverse reactions occurred in 85.7% (36/42) of patients and grade Ⅲ or Ⅳ adverse reactions occurred in 16.7% (7/42). There was no level Ⅳ adverse reactions. All adverse reactions were controlled after dose reduction and symptomatic treatment. Thirteen patients (31.0%, 13/42) redeemed resectable after treatment and underwent radical surgery. Seven patients had pathological CR after surgery. In two patients, the pathological residual cancer tissue was less than 10%. The cumulative overall survival rates of the 42 patients at 6 months, 1 year, 1.5 years after treatment were 100%, 91.7%, and 65.0%, respectively. The postoperative 1-year survival rate of patients undergoing surgery after successful conversion was 83.3%. Conclusion:This study preliminarily showed the safety and efficacy of TACE, TKI, and PD-1 inhibitor combined therapy in patients with initially unresectable hepatocellular carcinoma.

Objective To investigate the effect and its molecular mechanism of phosphoglycerate mutase 5 (PGAM5) mediated pyroptosis on liver ischemia-reperfusion injury (IRI). Methods C57 mouse models of liver IRI were established and randomly divided into the 6 h reperfusion (6 h group) and 12 h reperfusion (12 h group), and sham operation group (sham group) was established too, 10 rats in each group. The effect of IRI on the parameters in the liver tissues and serum samples was evaluated. The expression levels of PGAM5 and cysteinyl aspartate specific proteinase (Caspase)-1 in the liver tissues during IRI were quantitatively detected. The IRI models of liver cells were established (IRI group). The IRI models of liver cells were established after pretreatment with Caspase-1 inhibitor Z-YVAD-FMK (inhibitor group). The untreated AML12 cells were allocated into the control group. The effect of inhibiting Caspase-1 activity on pyroptosis was analyzed. AML12 cells were transfected with PGAM5 small interfering ribonucleic acid (siRNA) (siRNA group) and siRNA-negative control (siRNA-NC) (siRNA-NC group) by liposome 3000, and then IRI models of liver cells were established. The untreated AML12 cells were assigned into the control group. The effect of PGAM5 mediated pyroptosis on IRI of liver cells was assessed. Results In the 6 h and 12 h groups, partial liver cell edema, hepatic sinusoid narrowing, central vein congestion and occasional spot necrosis were observed in the mouse liver tissues, and these changes in the 12 h group were more aggravated than those in the 6 h group. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the 6 h and 12 h groups were higher than those in the sham group, and the values in the 12 h group were higher than those in the 6 h group. The levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β were increased in the 6 h and 12 h groups, and the values in the 12 h group were lower than those in the 6 h group. The relative expression levels of IL-1β messenger ribonucleic acid (mRNA) in the mouse liver tissues in the 6 h and 12 h groups were up-regulated, and the value in the 12 h group was lower than that in the 6 h group. The cell apoptosis rates in the liver tissues were significantly increased in the 6 h and 12 h groups, and the value in the 12 h group was remarkably lower than that in the 6 h group (P < 0.01-0.05). Compared with the sham group, the relative expression levels of PGAM5 mRNA and protein in the mouse liver tissues in the 6 h and 12 h groups were significantly up-regulated, and the values in the 12 h group were significantly higher than those in the 6 h group (P < 0.01-0.05). The protein expression levels of PGAM5 and Caspase-1 in the liver tissues were up-regulated in the 6 h and 12 h groups. Compared with the control group, the relative expression levels of NOD-like receptor protein 3 (NLRP3), cleaved Caspase-1 and Gasdermin D (GSDMD) proteins were up-regulated and the fluorescence intensity of GSDMD was increased in the IRI group. Compared with the IRI group, the relative expression levels of NLRP3, cleaved Caspase-1 and GSDMD proteins were significantly down-regulated and the fluorescence intensity of GSDMD was considerably decreased in the inhibitor group (P < 0.01-0.05). Compared with the control group, the cell survival rate was significantly decreased, and the relative expression levels of PGAM5, NLRP3, cleaved Caspase-1 and GSDMD proteins were significantly up-regulated in the siRNA-NC group (P < 0.01-0.05). Compared with the siRNA-NC group, the cell survival rate was remarkably increased, whereas the relative expression levels of PGAM5, NLRP3, cleaved Caspase-1 and GSDMD proteins were significantly down-regulated in the siRNA group (P < 0.01-0.05). Conclusions PGAM5 may aggravate the liver IRI in mouse models probably by mediating pyroptosis via PGAM5/Caspase-1/GSDMD signaling pathway and aggravating liver cell injury.