1.Correlation between gut microbiota and blood ammonia changes before and after TIPS in patients with decompensated cirrhosis
Dong ZHANG ; Meng HU ; Yongxiang ZHOU ; Dan LI ; Yuanjun DENG ; Guangjun TIAN
Journal of Interventional Radiology 2025;34(6):590-596
Objective To explore the effect of transjugular intrahepatic portosystemic shunt(TIPS)on gut microbiota and blood ammonia level in patients with decompensated cirrhosis,and to analyze the correlation between the gut microbiota and blood ammonia level.Methods From July 2021 to December 2023,the patients with decompensated cirrhosis and portal hypertension complicated by esophagogastric variceal bleeding and/or refractory ascites,who received TIPS at Zhuhai Hospital of Guangdong Provincial Hospital of Traditional Chinese Medicine,were collected.The blood and stool samples were collected before and after TIPS.The changes in blood routine,prothrombin time,liver function,and blood ammonia level were determined.The change of gut microbiota was detected using 16S rRNA high-throughput sequencing,and Spearman correlation analysis was used to assess the correlation between the gut microbiota and blood ammonia level.Results In 20 patients,the post-TIPS one-month levels of AST,TBil,DBiL,PT,and blood ammonia were higher than their pre-TIPS values(P<0.05),and the albumin(Alb)level was lower than the pre-TIPS value(P<0.05).The post-TIPS 3-month levels of TBil,DBil,PT,and blood ammonia were higher than their pre-TIPS values(P<0.05).One month after TIPS,the beta diversity of the gut microbiota became significantly different from the pre-TIPS pattern(P<0.05).After TIPS,the harmful bacteria such as Veillonella,Streptococcus and Haemophilus were significantly reduced,the difference was statistically significant(P<0.05).The correlation analysis of gut microbiota and blood ammonia level showed that in T0 group Colidextribacter was positively correlated with blood ammonia level,while Roxobella was negatively correlated with blood ammonia level;and in T1 group,Colidextribacter and Streptococcus were positively correlated with blood ammonia level,while Coprococcus,Bifidobacterium and Parasutterella were negatively correlated with blood ammonia level,and the differences were statistically significant(all P<0.05).Conclusion In patients with decompensated cirrhosis after receiving TIPS,significant changes in the pattern of gut microbiota occur.Certain correlations exist between the changes of some microbiota and blood ammonia levels.Regulating the intestinal microecology may contribute to reducing blood ammonia level after TIPS.
2.A learning curve analysis of domestic robot-assisted thyroid surgery via BABA approach
Jing XU ; Peng ZHOU ; Yongxiang LIU ; Jian ZHU ; Meng WANG ; Gang WANG ; Dayong ZHUANG ; Qingqing HE
Chinese Journal of Endocrine Surgery 2025;19(4):503-508
Objective:To summarise the main points of operation during thyroid surgery using bilateral axillo-breast approach (BABA) with a domestic robot and to analyse the learning curve.Methods:From May. 2023 to Oct. 2023, we retrospectively analysed the clinical data of 38 patients who underwent thyroid surgery with domestically manufactured robot via BABA attending the Department of Thyroid and Breast Surgery of the 960th Hospital of PLA Joint Logistics Support Force, proposed and constructed learning curves using cumulative and analytical methods to study the minimum number of learning cases required for the loading time of assistants and surgeon-in-charge with da Vinci surgical experience to transition from the initial learning stage to the proficiency stage. The age, BMI, surgical time (loading time, operating time), postoperative drainage time, and number of lymph nodes dissected were compared among patients at different stages of the learning curve.Results:A total of 38 thyroidectomy patients successfully underwent surgery, with an average surgical time of (176.61 ± 47.98) minutes. After 18 cases, the assistant's loading time shortened and stabilized at (42.20 ± 3.44) minutes. The operator reached peak proficiency at the 18th case (loading time, operating time), which was considered the dividing point between the learning phase and the proficiency phase. During the learning phase, the average surgical time was (209.72 ± 49.28) minutes, operation time: (165.44 ± 49.93) minutes, while in the proficiency phase, the average surgical time decreased to 146.80 ± 18.34 minutes, operation time: (104.60 ± 19.01) minutes. There were no statistically significant differences in baseline characteristics such as age, BMI, postoperative drainage time, or the number of central compartment lymph nodes dissected between the two phases ( P > 0.05) . Conclusion:Over the results of this study, it was shown that for robosurgeons with experience in da Vinci robotic surgery, the transition from the learning stage to the proficiency stage could be considered after crossing the learning curve for 18 cases, and that assistants could become proficient in loading techniques after 18 procedures.
3.Inhibition of tumor necrosis factor receptor associated factor 6 regulates mTORC1/ULK1 signaling and promotes autophagy to improve myocardial injury in sepsis mice
Ying ZHOU ; Yong TIAN ; Zhimei ZHONG ; Yongxiang GU ; Hao FANG
Chinese Journal of Tissue Engineering Research 2025;29(30):6434-6440
BACKGROUND:Studies have found that inhibition of tumor necrosis factor receptor-associated factor 6 improves myocardial function and promotes myocardial autophagy in sepsis,but the specific mechanism is not clear.OBJECTIVE:To explore the effect of inhibiting tumor necrosis factor receptor-associated factor 6-regulated mTORC1/ULK1 autophagy signaling pathway on myocardial injury in sepsis mice.METHODS:Thirty male Kunming mice were randomly divided into sham operation group,cecal ligation and puncture group(model group),model+tumor necrosis factor receptor-associated factor 6 specific inhibitor C25-140(model+C)group,model+C25-140+autophagy inhibitor 3-methyladenine(model+C+3-MA)group,and model+C25-140+mTORC1-specific agonist MHY1485(model+C+M)group.The cecum of mice in the sham operation group was not ligated or punctured.The mice in the other groups underwent cecum ligation and puncture to establish the mouse sepsis model.C25-140,3-methyladenine,and MHY1485 were intraperitoneally injected 0.5 hours after surgery according to the grouping.Myocardial tissue was obtained 24 hours after surgery.Hematoxylin-eosin staining was used to evaluate myocardial inflammatory lesions.Transmission electron microscopy was used to observe the changes in the autophagic bodies and mitochondrial microstructures of myocardial cells.TUNEL assay was used to detect myocardial cell apoptosis.PCR was used to detect the relative expression of tumor necrosis factor receptor-associated factor 6 mRNA.Western blot assay was used to detect the expression of related proteins.RESULTS AND CONCLUSION:(1)Compared with sham operation group,myocardial inflammatory cell infiltration and fibrous edema were observed in the model group.The mitochondria of the cells were obviously swollen,and autophagosomes were occasionally seen;cardiomyocyte apoptosis increased significantly;the expression of tumor necrosis factor receptor-associated factor 6,phosphorylated nuclear factor κB P65/P65,p-mTOR/mTOR,p-ULK1/ULK1,P62 and Bax protein increased,and the expression of Bcl2 protein decreased(P<0.05).(2)Compared with the model group,myocardial inflammation and fibrous edema were alleviated in the model+C group.Myocardial mitochondrial swelling was reduced and autophagosomes increased;cardiomyocyte apoptosis decreased;the expression of phosphorylated nuclear factor κB P65/nuclear factor-κB P65,p-mTOR/mTOR,p-ULK1/ULK1,P62,and Bax protein decreased,while the Beclin-1 and Bcl2 protein increased(P<0.05).(3)Compared with the model+C group,myocardial autophagosomes decreased and myocardial mitochondrial swelling was more obvious in the model+C+3-MA group.Myocardial inflammation was aggravated;myocardial cell apoptosis increased;the expression of phosphorylated nuclear factor κB P65/nuclear factor κB P65,P62,and Bax protein increased,and the Beclin-1 and Bcl2 protein decreased(P<0.05).(4)Compared with the model+C group,the expression of p-mTOR/mTOR and p-ULK1/ULK1 in the model+C+M group increased,and the Beclin-1 and microtubule-associated protein 1 light chain 3 Ⅱ/Ⅰ protein expression decreased(P<0.05).It is concluded that inhibition of tumor necrosis factor receptor-associated factor 6 regulates mTORC1/ULK1 autophagy signal to promote myocardial autophagy and participate in the protection of myocardial injury in sepsis.
4.A learning curve analysis of domestic robot-assisted thyroid surgery via BABA approach
Jing XU ; Peng ZHOU ; Yongxiang LIU ; Jian ZHU ; Meng WANG ; Gang WANG ; Dayong ZHUANG ; Qingqing HE
Chinese Journal of Endocrine Surgery 2025;19(4):503-508
Objective:To summarise the main points of operation during thyroid surgery using bilateral axillo-breast approach (BABA) with a domestic robot and to analyse the learning curve.Methods:From May. 2023 to Oct. 2023, we retrospectively analysed the clinical data of 38 patients who underwent thyroid surgery with domestically manufactured robot via BABA attending the Department of Thyroid and Breast Surgery of the 960th Hospital of PLA Joint Logistics Support Force, proposed and constructed learning curves using cumulative and analytical methods to study the minimum number of learning cases required for the loading time of assistants and surgeon-in-charge with da Vinci surgical experience to transition from the initial learning stage to the proficiency stage. The age, BMI, surgical time (loading time, operating time), postoperative drainage time, and number of lymph nodes dissected were compared among patients at different stages of the learning curve.Results:A total of 38 thyroidectomy patients successfully underwent surgery, with an average surgical time of (176.61 ± 47.98) minutes. After 18 cases, the assistant's loading time shortened and stabilized at (42.20 ± 3.44) minutes. The operator reached peak proficiency at the 18th case (loading time, operating time), which was considered the dividing point between the learning phase and the proficiency phase. During the learning phase, the average surgical time was (209.72 ± 49.28) minutes, operation time: (165.44 ± 49.93) minutes, while in the proficiency phase, the average surgical time decreased to 146.80 ± 18.34 minutes, operation time: (104.60 ± 19.01) minutes. There were no statistically significant differences in baseline characteristics such as age, BMI, postoperative drainage time, or the number of central compartment lymph nodes dissected between the two phases ( P > 0.05) . Conclusion:Over the results of this study, it was shown that for robosurgeons with experience in da Vinci robotic surgery, the transition from the learning stage to the proficiency stage could be considered after crossing the learning curve for 18 cases, and that assistants could become proficient in loading techniques after 18 procedures.
5.Inhibition of tumor necrosis factor receptor associated factor 6 regulates mTORC1/ULK1 signaling and promotes autophagy to improve myocardial injury in sepsis mice
Ying ZHOU ; Yong TIAN ; Zhimei ZHONG ; Yongxiang GU ; Hao FANG
Chinese Journal of Tissue Engineering Research 2025;29(30):6434-6440
BACKGROUND:Studies have found that inhibition of tumor necrosis factor receptor-associated factor 6 improves myocardial function and promotes myocardial autophagy in sepsis,but the specific mechanism is not clear.OBJECTIVE:To explore the effect of inhibiting tumor necrosis factor receptor-associated factor 6-regulated mTORC1/ULK1 autophagy signaling pathway on myocardial injury in sepsis mice.METHODS:Thirty male Kunming mice were randomly divided into sham operation group,cecal ligation and puncture group(model group),model+tumor necrosis factor receptor-associated factor 6 specific inhibitor C25-140(model+C)group,model+C25-140+autophagy inhibitor 3-methyladenine(model+C+3-MA)group,and model+C25-140+mTORC1-specific agonist MHY1485(model+C+M)group.The cecum of mice in the sham operation group was not ligated or punctured.The mice in the other groups underwent cecum ligation and puncture to establish the mouse sepsis model.C25-140,3-methyladenine,and MHY1485 were intraperitoneally injected 0.5 hours after surgery according to the grouping.Myocardial tissue was obtained 24 hours after surgery.Hematoxylin-eosin staining was used to evaluate myocardial inflammatory lesions.Transmission electron microscopy was used to observe the changes in the autophagic bodies and mitochondrial microstructures of myocardial cells.TUNEL assay was used to detect myocardial cell apoptosis.PCR was used to detect the relative expression of tumor necrosis factor receptor-associated factor 6 mRNA.Western blot assay was used to detect the expression of related proteins.RESULTS AND CONCLUSION:(1)Compared with sham operation group,myocardial inflammatory cell infiltration and fibrous edema were observed in the model group.The mitochondria of the cells were obviously swollen,and autophagosomes were occasionally seen;cardiomyocyte apoptosis increased significantly;the expression of tumor necrosis factor receptor-associated factor 6,phosphorylated nuclear factor κB P65/P65,p-mTOR/mTOR,p-ULK1/ULK1,P62 and Bax protein increased,and the expression of Bcl2 protein decreased(P<0.05).(2)Compared with the model group,myocardial inflammation and fibrous edema were alleviated in the model+C group.Myocardial mitochondrial swelling was reduced and autophagosomes increased;cardiomyocyte apoptosis decreased;the expression of phosphorylated nuclear factor κB P65/nuclear factor-κB P65,p-mTOR/mTOR,p-ULK1/ULK1,P62,and Bax protein decreased,while the Beclin-1 and Bcl2 protein increased(P<0.05).(3)Compared with the model+C group,myocardial autophagosomes decreased and myocardial mitochondrial swelling was more obvious in the model+C+3-MA group.Myocardial inflammation was aggravated;myocardial cell apoptosis increased;the expression of phosphorylated nuclear factor κB P65/nuclear factor κB P65,P62,and Bax protein increased,and the Beclin-1 and Bcl2 protein decreased(P<0.05).(4)Compared with the model+C group,the expression of p-mTOR/mTOR and p-ULK1/ULK1 in the model+C+M group increased,and the Beclin-1 and microtubule-associated protein 1 light chain 3 Ⅱ/Ⅰ protein expression decreased(P<0.05).It is concluded that inhibition of tumor necrosis factor receptor-associated factor 6 regulates mTORC1/ULK1 autophagy signal to promote myocardial autophagy and participate in the protection of myocardial injury in sepsis.
6.Pathophysiological characteristics of mice with diabetes combined with SARS-CoV-2 spike protein infection
Xiaoyue SU ; Jingxuan LI ; Ying LIN ; Yongxiang ZHANG ; Zhiyong XIAO ; Wenxia ZHOU
Chinese Journal of Pharmacology and Toxicology 2024;38(6):410-419
OBJECTIVE To establish a mouse model of diabetes mellitus(DM)combined with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection to investigate the important pathophysiological changes in the development of DM combined with SARS-CoV-2 infection.METHODS Wild-type(WT)mice and transgenic mice expressing the human angiotensin-converting enzyme 2 receptor driven by the cytokeratin-18 gene promoter(K18-hACE2)were randomly divided into the control group,DM group,SARS-CoV-2 spike protein(S)infection group and DM combined with S protein infection group,with 10 to 12 mice in each group.All the mice were induced by 10 weeks of high-fat diet combined with 40 mg·kg-1 streptozotocin(STZ)for 3 days by ip,except those in the control group or S protein infection group.The control group was given the same volume of 0.1 mol·L-1 sodium citrate buffer.Mice in the S protein infection group and DM+S protein infection group were additionally given 50 μL mixture of 15 μg SARS-CoV-2 spike protein and 1 g·L-1 polyinosinic-polycytidylic acid(poly[I:C])via intranasal drops,while the control group was given an equal volume of sterile water.The glucose tolerance level and pancreatic islet β cell function of mice were evaluated via oral glucose tolerance test at the 6th week of high-fat feeding and 1 week after the administration of STZ by ip.From the 6th week of high-fat feeding to 2 weeks after the administration of STZ,the random blood glucose and fasting blood glucose of mice were measured by a blood glucose meter.Blood samples were taken from subman-dibular veins of 3 mice in each group at 24,48 and 120 h after S protein infection,and lung tissues were taken after euthanization.The pathological changes of lungs of DM mice before and after S protein infection were observed by HE staining.Except for the DM group,blood samples were collected before S protein infection and at 6,24,48,72 and 120 h after infection.The levels of plasma interleukin 1β(IL-1β),IL-2,IL-6,IL-10,IL-17,interferon gamma-induced protein 10(IP-10),interferon γ(IFN-γ),tumor necrosis factor α(TNF-α),monocyte chemotactic protein-1(MCP-1)and granulocyte-colony stimulating factor(G-CSF)were detected by Luminex.The plasma levels of heparan sulfate(HS)were measured by enzyme-linked immunosorbent assay.The levels of cytokines and HS were correlated with the degree of pathological damage by Spearman correlation analysis.RESULTS STZ and high-fat diet could induce DM-like expression in mice,and the random blood glucose(P<0.01)and fasting blood glucose(P<0.05)after 1 week in the hACE2-DM group were significantly higher than in the WT-DM group,and the degree of islet function damage in hACE2-DM mice was significantly higher than that of WT-DM mice(P<0.05).Compared with the DM group,the DM+S group showed more severe pulmonary pathological changes after S protein infection,accompanied by a large number of inflammatory infiltrations and thickening of lung interstitial.Compared with the control group,the levels of pro-inflammatory cytokines G-CSF,IL-6 and IP-10 in the plasma of the WT-S group were significantly increased at 6 h after S pro-tein infection(P<0.01),and those of pro-inflammatory cytokine IL-17 and anti-inflammatory cytokine IL-10 were significantly increased at 24 h after S protein infection(P<0.05).Compared with the control group,the plasma levels of pro-inflammatory cytokines IL-1β,IL-6,TNF-α,MCP-1,G-CSF and IP-10 in the hACE2-S group were significantly increased at 6 h after S protein infection(P<0.05,P<0.01).IL-17 was significantly increased at 24 h and 6 h after S protein infection in the WT-DM+S group and hACE2-DM+S group,respectively(P<0.01,P<0.05).In the hACE2-DM+S group,IFN-γ and IL-1β were signifi-cantly increased in delay to 48 h(P<0.05,P<0.01),and MCP-1 was significantly increased in delay to 72h(P<0.05).Compared with the control group,the level of HS in the plasma of the WT-S group increased significantly(P<0.05,P<0.01)at 6 h and 24 h after S protein infection,but began to decrease at 48 h.At the same time,compared with the WT-S group,the HS level in the WT-DM+S group was slightly increased at 6 h after infection and decreased at 24 h.Compared with the control group,the HS level in the hACE2-S group was significantly increased at 24 h(P<0.01),as was the case with the WT-S group 24 h,48 h and 120 h after S protein infection.At 6 h,24 h and 48 h after S protein infection,the plasma HS level of the hACE2-DM+S group was significantly increased(P<0.01,P<0.05),and the duration of the increase was longer than in the hACE2-S group.Moreover,the levels of IL-1β,IL-10,MCP-1,IP-10,G-CSF and HS in plasma were positively correlated with the degree of lung dam-age in the DM+S group.CONCLUSION In this study,the mouse model of diabetes combined with SARS-CoV-2 spike protein infection has mimicked part of the pathophysiological features of clinical patients,mainly manifested as blunted immune response and elevated HS levels with longer duration to infection alone.IL-1β,IL-10,MCP-1,IP-10,G-CSF and HS may keep track of the course of disease in patients with diabetes combined with SARS-CoV-2 infection.
7.Damage effect and mechanisms of cyclophosphamide to human neuroblastoma SH-SY5Y cells
Jiajia LI ; Jiao WANG ; Wenyi XIAO ; Donghui WEI ; Yongxiang ZHANG ; Ning JIANG ; Wenxia ZHOU
Chinese Journal of Pharmacology and Toxicology 2024;38(8):561-574
OBJECTIVE To investigate the damage effect and mechanisms of cyclophosphamide(CTX)and its active metabolite derivative 4-hydroperoxycyclophosphamide(4-HC)to human neuroblas-toma SH-SY5Y cells.METHODS SH-SY5Y cells were treated with CTX[0(cell control),0.01,0.1,1,5,10,20,40 and 80 mmol·L-1]and 4-HC[0(cell control),0.01,0.1,1,5,10,20,40 and 80 μmol·L-1]for 48 h.Cell confluence and morphology were observed by the IncuCyte ZOOM system.Cell viability was assessed by CCK-8 assay.Lactate dehydrogenase(LDH)release was measured by LDH assay kit.SH-SY5Y cells were treated with CTX(0,1,5,10 and 20 mmol·L-1)and 4-HC(0,1,5,10 and 20 μmol·L-1)for 48 h before cell proliferation was analyzed by 5-ethynyl-2′-deoxyuridine(EdU)staining assay.Immunofluorescence was employed to assess the levels of the DNA double-strand break marker γ-H2AX and to evaluate changes in mitochondrial membrane potential.SH-SY5Y cells were treated with CTX(0,1,5 and 10 mmol·L-1)and 4-HC(0,1,5 and 10 μmol·L-1)for 48 h,and the alterations in glycolysis and oxidative phosphorylation levels were analyzed using the Seahorse XFe96 Analyzer.RESULTS Compared with the cell control group,cell confluence and cell viability were significantly reduced in the CTX and 4-HC groups(P<0.01),and the half-maximal inhibitory concentrations(IC50)for CTX and 4-HC were 4.44 mmol·L-1 and 4.78 μmol·L-1,respectively.The release rate of LDH was signif-icantly increased while the percentage of EdU+cells was significantly reduced in the CTX and 4-HC groups(P<0.01).The percentage of γ-H2AX+cells was significantly increased and mitochondrial membrane potential significantly decreased in the CTX and 4-HC group(P<0.05).Treatment with CTX and 4-HC resulted in reduced levels of maximum glycolytic capacity,glycolytic reserve,maximal respi-ration,and ATP production(P<0.05).CONCLUSION CTX and 4-HC exert significant cytotoxic effects on SH-SY5Y cells by disrupting cell membrane structure,impeding cell proliferation,and reducing cell viability.The mechanisms underlying these effects may involve intracellular DNA damage,disturbance of energy metabolism and mitochondrial dysfunction.
8.Clinical guidelines for the diagnosis and treatment of osteoporotic thoracolumbar vertebral fracture with kyphotic deformity in the elderly (version 2024)
Jian CHEN ; Qingqing LI ; Jun GU ; Zhiyi HU ; Shujie ZHAO ; Zhenfei HUANG ; Tao JIANG ; Wei ZHOU ; Xiaojian CAO ; Yongxin REN ; Weihua CAI ; Lipeng YU ; Tao SUI ; Qian WANG ; Pengyu TANG ; Mengyuan WU ; Weihu MA ; Xuhua LU ; Hongjian LIU ; Zhongmin ZHANG ; Xiaozhong ZHOU ; Baorong HE ; Kainan LI ; Tengbo YU ; Xiaodong GUO ; Yongxiang WANG ; Yong HAI ; Jiangang SHI ; Baoshan XU ; Weishi LI ; Jinglong YAN ; Guangzhi NING ; Yongfei GUO ; Zhijun QIAO ; Feng ZHANG ; Fubing WANG ; Fuyang CHEN ; Yan JIA ; Xiaohua ZHOU ; Yuhui PENG ; Jin FAN ; Guoyong YIN
Chinese Journal of Trauma 2024;40(11):961-973
The incidence of osteoporotic thoracolumbar vertebral fracture (OTLVF) in the elderly is gradually increasing. The kyphotic deformity caused by various factors has become an important characteristic of OTLVF and has received increasing attention. Its clinical manifestations include pain, delayed nerve damage, sagittal imbalance, etc. Currently, the definition and diagnosis of OTLVF with kyphotic deformity in the elderly are still unclear. Although there are many treatment options, they are controversial. Existing guidelines or consensuses pay little attention to this type of fracture with kyphotic deformity. To this end, the Lumbar Education Working Group of the Spine Branch of the Chinese Medicine Education Association and Editorial Committee of Chinese Journal of Trauma organized the experts in the relevant fields to jointly develop Clinical guidelines for the diagnosis and treatment of osteoporotic thoracolumbar vertebral fractures with kyphotic deformity in the elderly ( version 2024), based on evidence-based medical advancements and the principles of scientificity, practicality, and advanced nature, which provided 18 recommendations to standardize the clinical diagnosis and treatment.
9.Protective mechanism of metformin-induced cardiomyocyte autophagy on myocardial injury in septic mice
Yong Tian ; Ying Zhou ; Yongxiang Gu ; Guohui Yang
Acta Universitatis Medicinalis Anhui 2024;59(1):92-98
Objective :
To investigate the possible mechanism of metformin (Met) -induced cardiomyocyte autoph- agy in protecting myocardial injury in septic mice.
Methods :
The model of myocardial injury in septic mice was es- tablished by cecal ligation and puncture ( CLP) .Sixty Kunming mice were randomly divided into sham operation group (Sham group) ,model group ( CLP group) ,model + dimethyl sulfoxide ( DMSO) group ( CLP + DMSO group) ,model + metformin (Met) group (Met group) ,model + Met + 3-methyladenine (3-MA) group (Met + 3- MA group) ,model + Met + compound C ( CC) group (Met + CC group) ,with 10 mice in each group.The Met, Met + 3-MA and Met + CC groups were intraperitoneally injected with Met (200 mg / kg) once a day for 2 weeks be- fore modeling.The Met + 3-MA group was intraperitoneally injected with 3-MA ( 10 mg / kg) 1 h before surgery. The Met + CC group was intraperitoneally injected with CC (20 mg / kg) 30 min before surgery.The model was es- tablished 24 h after the last injection of Met.The heart and blood of all mice were collected 24 h after surgery.The Western blot technique was employed to assess the relative expression levels of microtubule-associated protein 1 light chain 3 (LC3) isoforms,namely LC3 I and LC3 II,autophagy effector protein 1 (Beclin-1) ,ubiquitin-bind- ing protein 62 (p62) ,B-cell lymphoma / leukemia-2 (Bcl-2) ,Bcl-2-associated X protein (Bax) ,adenosine mono- phosphate (AMP) kinase (AMPK) and phosphorylated AMPK (p-AMPK) .Myocardial pathological changes were observed by hematoxylin-eosin (HE) staining.The changes of myocardial mitochondria and autophagosomes were observed by electron microscopy.Hematoxylin-eosin (HE) staining was used to observe the pathological changes of myocardium. Electron microscopy was used to observe the changes of myocardial mitochondria and autophago- somes.
Results :
Compared with Sham group,the relative protein expression of Beclin-1,p62,p-AMPK / AMPK and LC3 II / LC3 I in CLP and CLP + DMSO groups had no statistical significance,but Bax increased and Bcl-2 de- creased in CLP group (P<0. 01) .Compared with CLP group,the relative expression of Beclin-1 protein and LC3 II / LC3 I in Met group increased and p62 decreased (P<0. 01) ,Bax decreased and Bcl-2 increased (P<0. 01) . Compared with Met group,the relative protein expression of Beclin-1 and LC3 II / LC3 I in Met + 3-MA group de- creased and p62 increased (P<0. 05) ,Bax increased and Bcl-2 decreased (P<0. 05) .Besides,the relative pro- tein expression of p-AMPK / AMPK in Met + CC group decreased (P<0. 05) .HE staining showed that there was no disorder in myocardial fibers in Sham group,and a large number of inflammatory cells infiltrated the myocardial fibers of CLP group in a clear disorder.The Met group showed vacuolar changes in the myocardium,while the Met + 3-MA group showed disordered arrangement of myocardial fibers and a small amount of inflammatory cell infiltra- tion.Under electron microscopy,the morphology of myocardial mitochondria in the Sham group was normal,while in the CLP group,the arrangement of mitochondrial cristae was disordered with vacuolar changes,and occasional autophagosomes were observed.Mitochondria in Met group showed slight swelling and a large number of autophago- somes.The mitochondria in the Met + 3-MA group showed significant swelling with a small amount of autophago- somes.
Conclusion
The protective effect of metformin on myocardial injury in septic mice can reduce cardiomyo- cyte apoptosis and improve mitochondrial damage by activating AMPK signaling pathway to induce autophagy.
10.Inhibition of TRAF6 ameliorates myocardial inflammatory injury and cardiac dysfunction via regulating cardiomyocyte inflammation in sepsis mice
Ying ZHOU ; Dajun JIANG ; Yong TIAN ; Yongxiang GU ; Guohui YANG
The Journal of Practical Medicine 2024;40(5):608-614
Objective To explore the effects of TRAF6 inhibition on autophagy,myocardial inflammation and cardiac function in septic mice.Methods Twenty-four male Kunming mice were randomly divided into 4 groups:sham,sham + C25-140(sham+C),cecal ligation and puncture(CLP),and cecal ligation and puncture+C25-140(CLP+C)group.Sham+C group and CLP+C group were intraperitoneally injected with C25-140 after operation.LVEF and LVFS were evaluated by ultrasound 24 hours after operation.Serum TNF-α and IL1-β were measured by ELISA.HE staining was used to evaluate myocardial inflammatory response.Autophagosomes and mitochondrial microstructure of cardiomyocytes were observed by transmission electron microscopy.TRAF6 mRNA in myocardial tissue was detected by qPCR.The expression of TRAF6,P62,Beclin-1 and LC3B protein was detected by W-B.The effect of C25-140 on myocardial injury in the septic mice was observed by inhibiting autophagy with 3-MA.Results Compared with the sham group,the levels of TRAF6 mRNA and TRAF6 in the myocardial tissue in the CLP group were significantly increased(P<0.05)and the serum TNF-α and IL1-β concentrations were signifi-cantly increased(P<0.05).Meanwhile,the myocardial tissue HE staining showed inflammatory cell infiltration and the LVEF and LVFS levels were significantly decreased in the CLP group(P<0.05).Compared with CLP group,the CLP+C group showed that the expression of TRAF6 mRNA and TRAF6 protein decreased(P<0.05),serum TNF-α and IL1-β decreased(P<0.05),myocardial histopathological myocardial inflammatory cell infiltration decreased,the LVEF and LVFS levels increased(P<0.05).Electron microscopy showed that the mitochondrial swelling decreased,autophagosomes increased,expression of Beclin-1 and LC3Ⅱ/Ⅰ increased,and P62 expression decreased(P<0.05).As compared with CLP+C group,the CLP+C+3-MA group showed that obvious inflamma-tory cell infiltration in the myocardial pathology and the LVEF and LVFS levels decreased after 3-MA inhibited autophagy(P<0.05).Conclusion Inhibition of TRAF6 can not only ameliorate myocardial inflammatory injury and cardiac dysfunction in septic mice,but promote the involvment of cardiomyocyte autophagy in provention from sepsis-induced myocardial injury.


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