To comprehend the genetic evolutionary characteristics and biological properties of the H4N1 subtype avian influenza virus(AIV)in China,this study conducted whole genome sequen-cing,genetic evolutionary analysis,and pathogenicity test in BALB/c mice of a duck-derived H4N1 subtype AIV strain(DK/GX/E1424/20)isolated from the live poultry market in the southern re-gion in 2020.The results indicated that the cleavage site motif of the HA protein was PEKASR/GLF,which conformed to the characteristics of low pathogenic AIV.All the eight gene fragments were situated in the Eurasian lineage,and the homology of AIV-related genes of the H1N1,H3N8,H4N6,H6N1,and H10N8 subtypes isolated from wild waterfowl was the highest,representing a recombinant virus strain.Without prior adaptation,it replicated effectively in the lungs and turbi-nates of mice,with viral titers of 3.00 and 2.08 log10EID50/mL respectively,and induced weight loss in infected mice.This study suggested that this virus had significant genetic diversity and low pathogenicity in mice,posing a potential risk for mammalian infection.

AIM:This study explores whether curcumin(Cur)promotes mitophagy to attenuate nonalcoholic steatohepatitis(NASH)in mice,as well as the possible molecular mechanisms involved.METHODS:A high-fat and high-cholesterol diet was used to replicate the NASH mouse model.Thirty-two male C57BL/6J mice were randomly divided into normal control(NC)group,high-fat and high-cholesterol model(M)group,M+low-dose Cur(Cur-L)group,and M+high-dose Cur(Cur-H)group,with 8 mice in each group.The weight of 8 mice in each group was recorded weekly.After feeding for 18 weeks,the serum and liver of mice were collected.Serum levels of total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL-C),alanine aminotransferase(ALT),aspartate aminotransferase(AST),and tumor necrosis factors-α(TNF-α)were measured.Liver index was calculated,and steatosis,inflammation,and fibrosis of the liver were observed by HE and Masson staining.Western blot analysis was performed to detect the protein expression of mi-tophagy-related protein,TNF-α and α-SMA in the liver.(2)HepG2 cells were treated with oleic acid and cholesterol to replicate the hepatocyte injury model,which was divided into NC group,Cur group,M group,and M+Cur group.Small interfering RNA for PTEN-induced kinase 1(PINK1)knockdown was used to explore the relationship between PINK1-me-diated mitophagy and NASH.Compound C(CC)was used to inhibit AMP-activated protein kinase(AMPK)to explore the effect of the AMPK/silent information regulator 1(Sirt1)pathway on mitophagy.The lipid droplets of HepG2 cells were ob-served by oil red O staining,and the levels of TC,TG,LDL-C,ALT,and AST in cell suspension were detected.RE-SULTS:(1)Compared with M group,treatment with Cur significantly reduced the body weight,liver coefficient,and se-rum levels of TC,TG,LDL-C,ALT,AST,and TNF-α in NASH mice,while the steatosis and fibrosis in the liver were improved(P<0.05).(2)Different concentrations of Cur could increase or decrease the expression of mitophagy-related proteins in HepG2 cells in a concentration gradient.Compared with the M group,Cur reduced lipid droplets and de-creased TC,TG,LDL-C,ALT,and AST levels(P<0.05).(3)Compared with the NC group,the expression levels of mi-tophagy-related proteins in the liver of mice in the M group decreased,and the expression levels of TNF-α and α-SMA pro-teins increased.Different concentrations of Cur intervention promoted the increase of mitophagy-related proteins and the decrease of TNF-α and α-SMA proteins(P<0.05).(4)After Cur intervention,the expression levels of mitophagy-related proteins increased and the expression levels of in TNF-α and α-SMA levels decreased in HepG2 cells induced by oleic acid and cholesterol(P<0.05).(5)Compared with M group,oleic-acidand cholesterol-induced mitophagy function in HepG2 cells was decreased after PINK1 knockdown(P<0.05).After CC inhibited AMPK,Cur increased the expression of p-AMPK(P<0.01),Sirt1(P<0.01),peroxisome proliferator-activated receptor γ coactivator-1α(P>0.05),PINK1(P<0.01)and parkin(P<0.01)proteins to some extent.CONCLUSION:Treatment with Cur attenuates liver injury in NASH mice and reduces lipid accumulation in HepG2 cells induced by oleic acid and cholesterol,and the mechanism may be related to promotion of mitophagy,which may involve the AMPK/Sirt1 signaling pathway.

BACKGROUND:Previous studies have shown that Huosui Formula has a synergistic effect on the immune and hematopoietic regulation of patients with myelodysplastic syndrome,but the specific mechanism is not yet clear.OBJECTIVE:To explore the effect and mechanism of Huosui Formula on bone marrow hematopoiesis in rats with myelodysplastic syndrome.METHODS:A total of 70 SD rats were randomly divided into a normal control group(n=10),a model group(n=15),a western medicine group(n=15),a low-dose Huosui Formula group(n=15),and a high-dose Huosui Formula group(n=15).Except for the normal control group,the other four groups were injected with dimethyl benzanthracene via the tail vein to induce the establishment of rat myelodysplastic syndrome models.After modeling,the normal control group and the model group were given normal saline;the western medicine group was given thalidomide capsules 10 mg/kg and retinoic acid tablets 4 mg/kg,and the low-dose Huosui Formula group and the high-dose Huosui Formula group were given 1.5 and 6 g/kg Huosui Formula,respectively,by intragastric administration once a day for 28 consecutive days.Peripheral blood and femoral bone marrow tissue were collected to detect peripheral blood routine and bone marrow biopsy hematopoietic proliferation.Flow cytometry was used to detect T lymphocyte subsets and the expression of CTLA-4 and PD-1 on T lymphocytes.RESULTS AND CONCLUSION:(1)Compared with the normal control group,peripheral blood leukocyte,neutrophil,hemoglobin,platelet,and CD4+,CD4+/CD8+levels were decreased in the model group significantly(P＜0.05),while CD4+PD-1+,CD8+PD-1+,CD4+CTLA-4+,and CD8+CTLA-4+expressions were significantly upregulated(P＜0.05).(2)In all dosage groups,myelopoietic proliferation was increased compared with the model group,with no significant difference between the groups(P＞0.05).(3)Compared with the model group,leukocytes,hemoglobin,platelets,and CD4+,CD4+/CD8+were significantly elevated in the high-dose Huosui Formula group(P＜0.05),the expression of CD8+was significantly lower(P＜0.05),and the levels of CD4+PD-1+,CD8+PD-1+,CD4+CTLA-4+,and CD8+CTLA-4+were down-regulated but not statistically significant(P＞0.05).(4)The western medicine group and the high-dose Huosui Formula group showed similar efficacy.The improvement of each index in the high-dose Huosui Formula group was superior to that in the low-dose Huosui Formula group.These findings indicate that Huosui Formula can improve the bone marrow hematopoiesis in myelodysplastic syndrome model rats,increase the levels of CD4+,and CD4+/CD8+while down-regulate the expression levels of CD4+PD-1+,CD8+PD-1+,CD4+CTLA-4+,and CD8+CTLA-4+.These observations suggest a link to the negative immunoregulation mechanism.

BACKGROUND:Previous studies have shown that Huosui Formula has a synergistic effect on the immune and hematopoietic regulation of patients with myelodysplastic syndrome,but the specific mechanism is not yet clear.OBJECTIVE:To explore the effect and mechanism of Huosui Formula on bone marrow hematopoiesis in rats with myelodysplastic syndrome.METHODS:A total of 70 SD rats were randomly divided into a normal control group(n=10),a model group(n=15),a western medicine group(n=15),a low-dose Huosui Formula group(n=15),and a high-dose Huosui Formula group(n=15).Except for the normal control group,the other four groups were injected with dimethyl benzanthracene via the tail vein to induce the establishment of rat myelodysplastic syndrome models.After modeling,the normal control group and the model group were given normal saline;the western medicine group was given thalidomide capsules 10 mg/kg and retinoic acid tablets 4 mg/kg,and the low-dose Huosui Formula group and the high-dose Huosui Formula group were given 1.5 and 6 g/kg Huosui Formula,respectively,by intragastric administration once a day for 28 consecutive days.Peripheral blood and femoral bone marrow tissue were collected to detect peripheral blood routine and bone marrow biopsy hematopoietic proliferation.Flow cytometry was used to detect T lymphocyte subsets and the expression of CTLA-4 and PD-1 on T lymphocytes.RESULTS AND CONCLUSION:(1)Compared with the normal control group,peripheral blood leukocyte,neutrophil,hemoglobin,platelet,and CD4+,CD4+/CD8+levels were decreased in the model group significantly(P＜0.05),while CD4+PD-1+,CD8+PD-1+,CD4+CTLA-4+,and CD8+CTLA-4+expressions were significantly upregulated(P＜0.05).(2)In all dosage groups,myelopoietic proliferation was increased compared with the model group,with no significant difference between the groups(P＞0.05).(3)Compared with the model group,leukocytes,hemoglobin,platelets,and CD4+,CD4+/CD8+were significantly elevated in the high-dose Huosui Formula group(P＜0.05),the expression of CD8+was significantly lower(P＜0.05),and the levels of CD4+PD-1+,CD8+PD-1+,CD4+CTLA-4+,and CD8+CTLA-4+were down-regulated but not statistically significant(P＞0.05).(4)The western medicine group and the high-dose Huosui Formula group showed similar efficacy.The improvement of each index in the high-dose Huosui Formula group was superior to that in the low-dose Huosui Formula group.These findings indicate that Huosui Formula can improve the bone marrow hematopoiesis in myelodysplastic syndrome model rats,increase the levels of CD4+,and CD4+/CD8+while down-regulate the expression levels of CD4+PD-1+,CD8+PD-1+,CD4+CTLA-4+,and CD8+CTLA-4+.These observations suggest a link to the negative immunoregulation mechanism.

To comprehend the genetic evolutionary characteristics and biological properties of the H4N1 subtype avian influenza virus(AIV)in China,this study conducted whole genome sequen-cing,genetic evolutionary analysis,and pathogenicity test in BALB/c mice of a duck-derived H4N1 subtype AIV strain(DK/GX/E1424/20)isolated from the live poultry market in the southern re-gion in 2020.The results indicated that the cleavage site motif of the HA protein was PEKASR/GLF,which conformed to the characteristics of low pathogenic AIV.All the eight gene fragments were situated in the Eurasian lineage,and the homology of AIV-related genes of the H1N1,H3N8,H4N6,H6N1,and H10N8 subtypes isolated from wild waterfowl was the highest,representing a recombinant virus strain.Without prior adaptation,it replicated effectively in the lungs and turbi-nates of mice,with viral titers of 3.00 and 2.08 log10EID50/mL respectively,and induced weight loss in infected mice.This study suggested that this virus had significant genetic diversity and low pathogenicity in mice,posing a potential risk for mammalian infection.

AIM:This study explores whether curcumin(Cur)promotes mitophagy to attenuate nonalcoholic steatohepatitis(NASH)in mice,as well as the possible molecular mechanisms involved.METHODS:A high-fat and high-cholesterol diet was used to replicate the NASH mouse model.Thirty-two male C57BL/6J mice were randomly divided into normal control(NC)group,high-fat and high-cholesterol model(M)group,M+low-dose Cur(Cur-L)group,and M+high-dose Cur(Cur-H)group,with 8 mice in each group.The weight of 8 mice in each group was recorded weekly.After feeding for 18 weeks,the serum and liver of mice were collected.Serum levels of total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL-C),alanine aminotransferase(ALT),aspartate aminotransferase(AST),and tumor necrosis factors-α(TNF-α)were measured.Liver index was calculated,and steatosis,inflammation,and fibrosis of the liver were observed by HE and Masson staining.Western blot analysis was performed to detect the protein expression of mi-tophagy-related protein,TNF-α and α-SMA in the liver.(2)HepG2 cells were treated with oleic acid and cholesterol to replicate the hepatocyte injury model,which was divided into NC group,Cur group,M group,and M+Cur group.Small interfering RNA for PTEN-induced kinase 1(PINK1)knockdown was used to explore the relationship between PINK1-me-diated mitophagy and NASH.Compound C(CC)was used to inhibit AMP-activated protein kinase(AMPK)to explore the effect of the AMPK/silent information regulator 1(Sirt1)pathway on mitophagy.The lipid droplets of HepG2 cells were ob-served by oil red O staining,and the levels of TC,TG,LDL-C,ALT,and AST in cell suspension were detected.RE-SULTS:(1)Compared with M group,treatment with Cur significantly reduced the body weight,liver coefficient,and se-rum levels of TC,TG,LDL-C,ALT,AST,and TNF-α in NASH mice,while the steatosis and fibrosis in the liver were improved(P<0.05).(2)Different concentrations of Cur could increase or decrease the expression of mitophagy-related proteins in HepG2 cells in a concentration gradient.Compared with the M group,Cur reduced lipid droplets and de-creased TC,TG,LDL-C,ALT,and AST levels(P<0.05).(3)Compared with the NC group,the expression levels of mi-tophagy-related proteins in the liver of mice in the M group decreased,and the expression levels of TNF-α and α-SMA pro-teins increased.Different concentrations of Cur intervention promoted the increase of mitophagy-related proteins and the decrease of TNF-α and α-SMA proteins(P<0.05).(4)After Cur intervention,the expression levels of mitophagy-related proteins increased and the expression levels of in TNF-α and α-SMA levels decreased in HepG2 cells induced by oleic acid and cholesterol(P<0.05).(5)Compared with M group,oleic-acidand cholesterol-induced mitophagy function in HepG2 cells was decreased after PINK1 knockdown(P<0.05).After CC inhibited AMPK,Cur increased the expression of p-AMPK(P<0.01),Sirt1(P<0.01),peroxisome proliferator-activated receptor γ coactivator-1α(P>0.05),PINK1(P<0.01)and parkin(P<0.01)proteins to some extent.CONCLUSION:Treatment with Cur attenuates liver injury in NASH mice and reduces lipid accumulation in HepG2 cells induced by oleic acid and cholesterol,and the mechanism may be related to promotion of mitophagy,which may involve the AMPK/Sirt1 signaling pathway.

AIM:The aim of this study was to investigate the effects and mechanism of high fat on autolyso-somes in hepatoma cells before and after serum deprivation.METHODS:HepG2 cells were intervened with 1 mmol/L so-dium oleate to create a cell high-fat model.The gene expression of transcription factor EB(TFEB)in HepG2 cells was knocked down using TFEB small interfering RNA(TFEB-siRNA)transfection reagent.AMP-activated protein kinase(AMPK)inhibitor compound C(CC)was used to inhibit AMPK phosphorylation expression in HepG2 cells.The expres-sion of nuclear and cytoplasmic TFEB,lysosome-associated membrane protein 1(LAMP1),microtubule-associated pro-tein light chain 3(LC3),autophagy adaptor protein(p62),AMPK,and p-AMPK proteins in each group was analyzed through Western blot experiments.Lipid metabolism and liver function damage in each group were analyzed using total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C),alanine aminotransferase(ALT),and aspartate aminotransferase(AST)assay kits.The accumulation of lipid droplets in each group of cells was analyzed through oil red O staining.RESULTS:(1)Sodium oleate intervention resulted in a concentration-dependent decrease in the protein expression levels of LAMP1,LC3-Ⅱ/LC3-Ⅰ,and nuclear TFEB,while increasing the protein expression level of p62(P＜0.01).(2)Compared to the NC group,the sodium oleate group showed decreased expression of LAMP1,LC3-Ⅱ/LC3-Ⅰ,nuclear TFEB,and AMPK phosphorylation levels,with an increase in p62 expression.Compared to the sodium oleate group,the sodium oleate+serum deprivation combined intervention group showed increased nuclear TFEB,LAMP1,LC3-Ⅱ/LC3-Ⅰ,AMPK phosphorylation levels,and decreased p62 expression levels(P＜0.05).(3)The levels of TC,TG,LDL-C,ALT,and AST were increased in the sodium oleate group compared to the NC group.Serum deprivation reduced the number of lipid droplets induced by sodium oleate in HepG2 cells and decreased the levels of TC,TG,LDL-C,ALT,and AST compared to the sodium oleate group(P＜0.05).(4)Knockdown of TFEB did not result in significant changes in the levels of nuclear TFEB,LAMP1,LC3-Ⅱ/LC3-Ⅰ,p62,TC,TG,LDL-C,ALT and AST compared to the so-dium oleate group.(5)Inhibition of AMPK phosphorylation did not result in significant changes in the levels of nuclear TFEB,LAMP1,LC3-Ⅱ/LC3-Ⅰ,p62,and AMPK phosphorylation in the sodium oleate+serum deprivation group com-pared to the sodium oleate group.CONCLUSION:Serum deprivation improves sodium oleate-induced lipid metabolism damage in HepG2 cells through the autophagolysosome pathway mediated by AMPK-TFEB.

AIM:The aim of this study was to investigate the effects and mechanism of high fat on autolyso-somes in hepatoma cells before and after serum deprivation.METHODS:HepG2 cells were intervened with 1 mmol/L so-dium oleate to create a cell high-fat model.The gene expression of transcription factor EB(TFEB)in HepG2 cells was knocked down using TFEB small interfering RNA(TFEB-siRNA)transfection reagent.AMP-activated protein kinase(AMPK)inhibitor compound C(CC)was used to inhibit AMPK phosphorylation expression in HepG2 cells.The expres-sion of nuclear and cytoplasmic TFEB,lysosome-associated membrane protein 1(LAMP1),microtubule-associated pro-tein light chain 3(LC3),autophagy adaptor protein(p62),AMPK,and p-AMPK proteins in each group was analyzed through Western blot experiments.Lipid metabolism and liver function damage in each group were analyzed using total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C),alanine aminotransferase(ALT),and aspartate aminotransferase(AST)assay kits.The accumulation of lipid droplets in each group of cells was analyzed through oil red O staining.RESULTS:(1)Sodium oleate intervention resulted in a concentration-dependent decrease in the protein expression levels of LAMP1,LC3-Ⅱ/LC3-Ⅰ,and nuclear TFEB,while increasing the protein expression level of p62(P＜0.01).(2)Compared to the NC group,the sodium oleate group showed decreased expression of LAMP1,LC3-Ⅱ/LC3-Ⅰ,nuclear TFEB,and AMPK phosphorylation levels,with an increase in p62 expression.Compared to the sodium oleate group,the sodium oleate+serum deprivation combined intervention group showed increased nuclear TFEB,LAMP1,LC3-Ⅱ/LC3-Ⅰ,AMPK phosphorylation levels,and decreased p62 expression levels(P＜0.05).(3)The levels of TC,TG,LDL-C,ALT,and AST were increased in the sodium oleate group compared to the NC group.Serum deprivation reduced the number of lipid droplets induced by sodium oleate in HepG2 cells and decreased the levels of TC,TG,LDL-C,ALT,and AST compared to the sodium oleate group(P＜0.05).(4)Knockdown of TFEB did not result in significant changes in the levels of nuclear TFEB,LAMP1,LC3-Ⅱ/LC3-Ⅰ,p62,TC,TG,LDL-C,ALT and AST compared to the so-dium oleate group.(5)Inhibition of AMPK phosphorylation did not result in significant changes in the levels of nuclear TFEB,LAMP1,LC3-Ⅱ/LC3-Ⅰ,p62,and AMPK phosphorylation in the sodium oleate+serum deprivation group com-pared to the sodium oleate group.CONCLUSION:Serum deprivation improves sodium oleate-induced lipid metabolism damage in HepG2 cells through the autophagolysosome pathway mediated by AMPK-TFEB.

Objective:To investigate the effects of 30% ethanol elution fraction of Artemisia absinthium extract with macroporous resin (AAEM-30%) on the dendritic cell (DC) and immunity of mice. Methods:AAEM-30% was obtained from the alcoholic extracts of A. absinthium by AB-8 macroporous resin, and its polysaccharide, flavonoid, and terpenoid contents were determined. The expressions of AAEM-30% on DC surface molecular cluster of differentiation (CD) 40, CD80 and CD86 were detected in vitro by flow cytometry, and the expressions of DC cytokines IL-6 and tumor necrosis factor-α (TNF-α) were detected by enzyme linked immunosorbent assay (ELISA). The effect of AAEM-30% on the immune function of ICR mice was measured in vivo with different doses (50 and 100 mg/kg) and different administration methods (subcutaneous injection, intraperitoneal injection, and gavage). Results:The contents of polysaccharides, flavonoids, and terpenoids in AAEM-30% were 24.30%, 22.50% and 28.19%, respectively. AAEM-30% significantly enhanced the expression of CD40, and CD86 and the secretion of IL-6 and TNF-α (all P<0.001). Compared with the control group, no statistically significant differences were found in the body mass of mice compared with the three administration methods (all P>0.05). The thymus index in the 50 and 100 mg/kg AAEM-30% intraperitoneal injection groups and the spleen index in the 50 mg/kg AAEM-30% gavage group were increased (all P<0.05). CD19 + cells increased in the 100 mg/kg AAEM-30% intraperitoneal injection group ( P<0.01) and in the 50 mg/kg AAEM-30% gavage group ( P<0.05). The CD11b + and CD11c + counts increased in the 100 mg/kg AAEM-30% gavage group ( P<0.05). The number of CD4 + and CD8 + T lymphocytes was increased by both gavage and intraperitoneal administration (all P<0.05). Conclusions:AAEM-30% can promote the maturation of DC and enhanced the immunity of mice without obvious side effects.

Objective:To investigate the efficacy of endoscopic histoacryl injection in cirrhotic patients with newly-developed esophagogastric varices (EGV) who have previously undergone splenectomy combined with pericardial devascularization.Methods:From January 2015 to January 2020, 125 cirrhotic patients with EGV treated with endoscopic histoacryl injection at the Department of Gastroenterology, Jinling Hospital, Medical School of Nanjing University, were included in the retrospective analysis. There were 45 patients in the group of splenectomy combined with pericardial devascularization (splenectomy group for short) and 80 patients in the non-splenectomy group. The efficacy of endoscopic treatment, postoperative variceal improvement, rebleeding rate, and complications were analyzed between the two groups.Results:Endoscopic histoacryl injection was successfully completed in all 125 patients, and the median volume of histoacryl was 4.5 mL. The overall effective rate in splenectomy and non-splenectomy group was 80.0% (36/45) and 57.5% (46/80), respectively. The difference in the number of significantly effective, effective, and ineffective cases between the two groups was statistically significant (16, 20, 9 cases, and 20, 26, 34 cases, respectively, χ 2=6.469, P=0.039). Two and 14 patients developed rebleeding in the splenectomy group and non-splenectomy group, respectively; and the difference in the rebleeding rate between the two groups was statistically significant (4.4% VS 17.5%, Log-rank P=0.039). No patient died within 1 year in either group, and no serious complications such as ectopic embolism occurred. Conclusion:After splenectomy combined with pericardial devascularization in cirrhotic patients with EGV and hypersplenism, the application of histoacryl has better short-term efficacy and can significantly reduce the rebleeding rate compared with the non-splenectomy group.