1.Advance in the induction of islet-like cell from bone marrow mesenchymal stem cells through gene transfection
Sheng BAI ; Mingwei SHENG ; Xiaomian LI
International Journal of Biomedical Engineering 2011;34(4):232-234,239
As the progress of organ and tissue transplantation technology, pancreas and islet transplantation has been introduced to treat diabetes and became hot. However, many shortcomings and re-lated issues need to be improved concerning the islet cell sources. Bone marrow mesenchymal stem cells (BMSCs) has become the hot topic on induction of islet-like cell because it was simple to access, easy to culture and possess the function of immunosuppressian. In recent years, artificial construction of pancreatic islet cells, which uses gene recombinant and gene transfection to reforge islet-like cells, has improved the induction rate of BMSCs and increased the amount of insulin serection, and it also provide new ideas and direction for the treatment of diabetes.
2.In vitro study on Coxsackie virus B3 infection on islet cells
Mingwei SHENG ; Sisi CHEN ; Sheng BAI ; Yu ZHENG ; Xiaomian LI
Chinese Journal of Microbiology and Immunology 2011;(3):229-233
Objective To study the effect of Coxsackie virus B3 (CVB3) infection on islet cells in vitro, and to explore the mechanism of islet cells caused by CVB3. Methods Bone marrow mesenchymal stem cells( BMSCs) were separated from the bone marrow and cultured. Then they were induced to differentiate into islet-like cells using nicotinamide and mercaptoethanol. Differentiated cells were detected by morphology , special staining and RT-PCR. Observe CVB3 infection on islet cells under inverse microscope and detect the specific gene fragment by RT-PCR. Results BMSCs showed half suspended shape and gathered to form a cluster after induction. Cells became red brown by dithizone specific staining. RT-PCR also proved the existence of mRNA expressing insulin. Infected islet cells appeared typical pathological changes like shrinks, refraction decreases. RT-PCR detected the desired specific gene fragment of 299 bp in infected islet cells. Conclusion CVB3 can directly injury islet cells, and damage the function of islet cells of secreting insulin.
3.Berberine preconditioning protects against hepatic cold ischemia reperfusion injury through the modulation of PI3K/Akt/mTOR signaling pathway
Mingwei SHENG ; Yuan ZHOU ; Wenli YU ; Yiqi WENG ; Hongyin DU
Chinese Journal of Organ Transplantation 2015;36(1):34-39
Objective To confirm the protective effect of berberine (BBR) on cold ischemia reperfusion (I/R)-induced liver injury and to show whether the hepatic protection conferred by BBR involves the activation of phosphatidylinositol 3 kinase (PI3K) / protein kinase B (Akt)/mammalian target of rapamycin(mTOR) signal pathway.Method Adult male Sprague-Dawley rats were assigned randomly to four groups:BBR group (BBR was intragastrically administered at a dose of 100 mg·kg-1 · d-1 2 weeks before hepatic cold I/R treatment),dimethyl sulfoxide (DMSO) group (BBR was replaced by DMSO,and others were the same as BBR group),I/R group (BBR was replaced by normal saline,and others were the same as BBR group) and sham group (normal saline was administered 2 weeks before opening and closing abdomen treatment).Then the rats were sacrificed at 3,6,and 24 h after reperfusion.The liver function,oxidative stress level,apoptosis rate,and the expression of PI3K/Akt/mTOR related pathway proteins were assayed.Result As compared with sham group,the I/R-induced liver tissue displayed severe lobular distortion with widespread necrosis,high level of oxidative stress and apoptosis rate.As compared with I/R group,BBR dramatically attenuated the histopathologic damage,restored the liver function and decreased the oxidative stress level.Simultaneously,BBR significantly ameliorated the apoptosis by decreasing the apoptosis rate,increasing the Bcl-2/Bax ratio and inhibiting caspase-3 activity in rats subjected to hepatic I/R.The expression of p-Akt was effectively upregulated with the inhibited expression of p-mTOR.Conclusion Our result provides robust in vivo evidence that BBR can prevent I/R-induced oxidative stress and apoptosis.The mechanisms involved can be attributed to the activation of P]3K/Akt/mTOR signal pathway.
4.Effect of dexmedetomidine on myocardial injury in pediatric patients undergoing living-related liver transplantation
Mingwei SHENG ; Hongyin DU ; Wenli YU ; Yiqi WENG ; Ying SUN
Chinese Journal of Anesthesiology 2017;37(3):263-266
Objective To evaluate the effect of dexmedetomidine on myocardial injury in pediatric patients undergoing living-related liver transplantation. Methods Fifty-eight pediatric patients of both sexes,aged 5-20 months,weighing 4.5-15.0kg,of American Society of Anesthesiologists physical status Ⅲ or Ⅳ,scheduled for elective living-related liver transplantation,were divided into 2 groups(n=29 each)using a random number table:control group(group C)and dexmedetomidine group(group D).In group D,dexmedetomidine was intravenously infused at a dose of 0.5 μg/kg over 10min starting from the time point immediately before skin incision,followed by an infusion of 0.8 μg·kg-1·h-1 until the end of surgery. The equal volume of normal saline was given instead in group C. Immediately before skin incision(baseline,T0),at 10min of anhepatic phase(T1),at 30min of neohepatic phase(T2)and at the end of surgery(T3),blood samples were obtained from the central vein for determination of serum cardiac troponin I(cTnI),lactate dehydrogenase(LDH),alpha-hydroxybutyrate dehydrogenase(α-HBDH),interleukin-6(IL-6)and IL-10 concentrations. The changing rate of serum cTnI concentrations were calculated at T2. The occurrence of myocardial ischemia and ventricular premature beat and requirement for dopamine were recorded during surgery. Results Compared with the baseline at T0,the serum concentrations of cTnI,LDH and α-HBDH were significantly increased at T2,3,and the serum concentrations of IL-6 and IL-10 were increased at T1-3 in both groups(P<0.05).Compared with group C,the serum concentrations of cTnI,LDH,α-HBDH and IL-6 were significantly decreased at T2,3,the serum concentration of IL-10 was increased at T1-3,the changing rate of serum cTnI concentrations was decreased(P<0.05),and no significant change was found in the incidence of myocardial ischemia and ventricular premature beat and requirement for dopamine in group D(P>0.05).Conclusion Dexmedetomidine can attenuate the myocardial injury to some extent in pediatric patients undergoing living-related liver transplantation.
5.Role of SIRT1/FoxO3α signaling pathway in berberine pretreatment-induced reduction of hypoxia/ reoxygenation-caused injury to hepatic parenchymal cells
Mingwei SHENG ; Yuanbang LIN ; Hongyin DU ; Wenli YU ; Lili JIA
Chinese Journal of Anesthesiology 2017;37(4):504-507
Objective To evaluate the role of silent information regulator fac tor 2-related enzyme 1 (SIRT1)/Forkhead Box O3 (FoxO3a) signaling pathway in berberine pretreatment-induced reduction of hypoxia/reoxygenation (H/R)-caused injury to hepatic parenchymnal cells.Methods Hepatic parenchymal cells obtained from AML12 mice were cultured and seeded in 6-well plates (2 ml/well) and in 96-well plates (200 μl/well) at the density of l×l06 cells/ml.The cells were divided into 4 groups (n=36 each)using a randomn number table:control group (group C),group H/R,berberine pretreatment group (group BP) and SIRT1-siRNA group (group SS).The cells were cultured in normal culture atmosphere (5% CO2-21% O2-74% N2) in group C.In H/R,BP and SS groups,the cells were exposed to hypoxic air (5% CO2-1% O2-94% N2) for 12 h,followed by 6 h reoxygenation in normal culture atmosphere (5% CO2-21% O2-74% N2).In group SS,small interference RNA targeting SIRT1 (SIRT1-siRNA) was added to the culture medium at 24 h prior to hypoxia.Berberine (final concentration 5 μmol/L) was added at 2 h prior to hypoxia in BP and SS groups.At the end of reoxygenation,the cell viability was measured by methyl thiazolyl tetrazolium assay,the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined using enzyme-linked immunosorbent assay,cell apoptosis was detected by flow cytometry,the expression of SIRT1 and FoxO3α was detected by Western blot,and the acetylation of FoxO3α was measnred by using immunoprecipitation.Apoptotic rate was calculated.Results Compared with group C,the cell viability was significantly decreased,the MDA content was increased,the SOD activity was decreased,apoptotic rate was increased,the expression of SIRT1 and ratio of FoxO3α expression in nucleus/in cytoplasma were increased,and the acetylation of FoxO3α in the nucleus was increased in H/ R,BP and SS groups (P< 0.05).Compared with group H/R,the cell viability was significantly increased,the MDA content was decreased,the SOD activity was increased,apoptotic rate was decreased,the expression of SIRT1 and ratio of FoxO3α expression in nucleus/in cytoplasma were increased,and the acetylation of FoxO3α in the nucleus was increased in group BP (P<0.05).Compared with group BP,the cell viability was significantly decreased,the MDA content was increased,the SOD activity was decreased,apoptotic rate was increased,the expression of SIRT1 and ratio of FoxO3α expression in nucleus/in cytoplasma were decreased,and the acetylation of FoxO3α in the nucleus was decreased in group SS (P<O.05).Conclusion The mechanism by which berberine pretreatment attenuates H/R-caused injury to hepatic parenchymal cells is related to promotion of SIRT1 expression in cells and inhibition of FoxO3α acetylation in the nucleus.
6.Prognostic value of serum cardiac troponin Ⅰ in acute lung injury after living-donor liver transplantation for children with biliary atresia
Mingwei SHENG ; Hongyin DU ; Yiqi WENG ; Rubin XU ; Ying SUN ; Wenli YU
Chinese Journal of Organ Transplantation 2016;37(10):581-585
Objective To evaluate the impact of cardiac troponin Ⅰ (cTnI) on acute lung injury in pediatric living donor liver transplant children with biliary atresia.Methods The clinical data of 112 pediatric living donor liver transplant recipients with biliary atresia in Tianjin First Central Hospital from February 2011 to September 2015 were retrospectively reviewed.Fifty-five recipients with cTnI ≥0.07 μg/L served as high-cTnI group and 57 recipients with cTnI group <0.07μg/L as normalcTnI group.The clinical data between two groups were compared and the association between serum cTnI level and acute lung injury after living-donor liver transplantation was evaluated by logistic regression analysis.Results The percentage of acute lung injury after pediatric living donor liver transplantation in high-cTnI group and normal-cTnI group was 31.6% and 9.1%,respectively.Intratransplant cTnI ≥0.07μg/L (OR =4.489,confidence interval 1.170-17.226) was the risk factor for acute lung injury after transplantation.The value of cTnI showed the positive correlation with preoperative PELD scores (OR =4.489,confidence interval 1.170-17.226).Conclusions Intratransplant cTnI level was the significant prognostic risk factor in acute lung injury after pediatric living-donor liver transplantation for children with biliary atresia.The cTnI level was associated with preoperative PELD scores.
7.Role of autophagy in hydrogen-induced inhibition of apoptosis in hippocampal neurons in a rat model of orthotopic liver transplantation
Mingxin CHEN ; Hongyin DU ; Wenli YU ; Li WU ; Dongjing SHI ; Hengchang REN ; Mingwei SHENG ; Shusen WANG
Chinese Journal of Anesthesiology 2017;37(2):184-187
Objective To evaluate the role of autophagy in hydrogen-induced inhibition of apoptosis in hippocampal neurons in a rat model of orthotopic liver transplantation (OLT).Methods Fifty-six pathogen-free healthy adult male Sprague-Dawley rats,aged 8-10 weeks,weighing 220-250 g,were used in the study.Thirty-two rats were selected and assigned into 4 groups (n =8 each) using a random number table:sham operation group (group S),OLT group,hydrogen-rich saline group (group HS) and chloroquine group (group CQ).The other 24 rats severed as the donors.In group S,laparotomy was performed,and the related blood vessels were isolated.The model of OLT was established in OLT,HS and CQ groups.In group OLT,normal saline 6 ml/kg was slowly injected via the inferior vena cava at 5 min before anhepatic phase.In group HS,hydrogen-rich saline 6 ml/kg was slowly injected via the inferior vena cava at 5 min before anhepatic phase.In group CQ,autophagy inhibitor chloroquine 60 mg/kg was injected intraperitoneally at 1 h before establishment of the model,and the other treatments were similar to those previously described in group HS.At 6 h of reperfusion,the rats were sacrificed and hippocampi were isolated for determination of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity,for pathological examination (with light microscope),and for detection of cell apoptosis (by TUNEL staining) and expression of autophagy-and apoptosis-related proteins caspase-3,cytochrome c (Cyt c),microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ),Beclin-1 and p53 in hippocampal tissues (by Western blot analysis).Apoptosis index (AI) was calculated.Results Compared with group S,the MDA content and AI were significantly increased,the SOD activity was decreased,and the expression of caspase-3,Cyt c,LC3 Ⅱ,Beclin-1 and p53 was up-regulated in OLT,HS and CQ groups (P<0.05).Compared with group OLT,the MDA content and AI were significantly decreased,the SOD activity was increased,the expression of caspase-3 and Cyt c was down-regulated,and the expression of LC3 Ⅱ,Beclin-1 and p53 was up-regulated in group HS (P<0.05).Compared with group HS,the MDA content and AI were significantly increased,the SOD activity was decreased,and the expression of caspase-3 and Cyt c was up-regulated,and the expression of LC3 Ⅱ,Beclin-1 and p53 was down-regulated in group CQ (P<0.05).Conclusion The mechanism by which hydrogen inhibits apoptosis in hippocampal neurons is related to promotion of autophagy in a rat model of OLT.
8.Effect of dexmedetomidine on kidney injury induced by liver ischemia-reperfusion in rats
Lili JIA ; Fei WANG ; Yiqi WENG ; Wenli YU ; Mingwei SHENG ; Hongyin DU
Chinese Journal of Anesthesiology 2016;36(2):223-226
Objective To evaluate the effect of dexmedetomidine on kidney injury induced by liver ischemia-reperfusion (I/R) in rats.Methods Twenty-four healthy male Sprague-Dawley rats,weighing 220-250 g,aged 8-10 weeks,were randomly divided into 3 groups (n=8 each) using a random number table:sham operation group (group S);liver I/R group (group I/R);dexmedetomidine group (group D).In group I/R,liver I/R model was established by clamping the portal vein,hepatic artery,supra-and infra-hepatic vena cava for 40 min,followed by 6 h of reperfusion in anesthetized rats.In group D,dexmedetomidine 100 μg/kg was injected intraperitoneally at 30 min before skin incision.The equal volume of normal saline was given instead of dexmedetomidine in S and I/R groups.At 6 h of reperfusion,blood samples were collected from the infra-hepatic vena cava for determination of blood urea nitrogen (BUN) and creatinine (Cr) concentrations (by automatic biochemical analyzer) and tumor necrosis factor-alpha (TNFα) and interleukin-10 (IL-10) concentrations in serum (by enzyme-linked immunosorbent assay).After blood sampling,the rats were sacrificed,and kidneys were harvested for examination of histopathological changes (with light microscope) and for determination of malondialdehyde (MDA) content (using thiobarbituric acid method) and superoxide dismutase (SOD) activity (by xanthine oxidase method),expression of activated caspase-3 (by immuno-histochemistry),and apoptotic cells (using TUNEL).Apoptotic rate was calculated.Results Compared with group S,the serum BUN,Cr and TNF-α concentrations were significantly increased,the concentration of serum IL-10 was decreased,the MDA content and apoptotic rate were increased,the SOD activity was decreased,and the expression of activated caspase-3 was up-regulated in I/R and D groups (P<0.05).Compared with group I/R,the serum BUN,Cr and TNF-α concentrations were significantly decreased,the concentration of serum IL-10 was increased,MDA content and apoptotic rate were increased,the SOD activity was decreased,the expression of activated caspase-3 was down-regulated (P<0.05),and the histopathological changes of renal tissues were attenuated in group D.Conclusion Dexmedetomidine can reduce kidney injury induced by liver I/R in rats,and the mechanism is probably related to inhibition of inflammatory responses,lipid peroxidation and cell apoptosis.
9.Effect of berberine pretreatment on hypoxia/reoxygenation-induced apoptosis in human renal tubular epithelial cells
Mingwei SHENG ; Hongyin DU ; Wenli YU ; Yiqi WENG ; Rubin XU ; Hengchang REN
Chinese Journal of Anesthesiology 2013;(2):232-235
Objective To investigate the effect of berberine pretreatment on hypoxia/reoxygenation (H/R)-induced apoptosis in human renal tubular epithelial cells.Methods The human renal tubular epithelial cells were cultured and seeded in culture dishes (2 ml/dish) or 96-well plates (200 μl/well) with the density of 1 ×106/ml.The cells were then randomly divided into 4 groups (n =30 each):normal control group (group C),berberine group (group B),H/R group and H/R + berberine group (H/R + B group).In groups B and H/R + B,berberine 10 μmol/L was added to the culture medium and the cells were incubated for 2 h.Groups H/R and H/R + B were then exposed to 94% N2-5% CO2-1% O2 for 24 h followed by 3 h reoxygenation.The cell viability,apoptotic rate and level of malondialdehyde (MDA) and superoxide dismutase (SOD) were detected.The expression of caspase-3,activated caspase-3,cytochrome c,glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) was determined.The ratio of Bax/Bcl-2 was calculated.Results Compared with group C,the cell viability,SOD activity and caspase-3 expression were significantly decreased,the apoptotic rate,Bax/Bcl-2 ratio and concentration of MDA were increased,and the expression of activated caspase-3,cytochrome c,GRP78 and CHOP was up-regulated in groups H/R and H/R + B (P < 0.05).Compared with group H/R,the cell viability,SOD activity and caspase-3 expression were significantly increased,the apoptotic rate,Bax/Bcl-2 ratio and concentration of MDA were decreased,and the expression of activated caspase-3,cytochrome c,GRP78 and CHOP was down-regulated in group H/R + B (P < 0.05).Conclusion Berberine pretreatment can inhibit H/R-induced apoptosis in human renal tubular epithelial cells,and inhibition of mitochondrial stress pathway and endoplasmic reticulum stress pathway is involved in the mechanism.
10.Role of Nrf2/HO-1 signaling pathway in reduction of acute kidney injury following orthotopic liver transplantation by hydrogen-rich saline in rats
Li WU ; Hongyin DU ; Wenli YU ; Dongjing SHI ; Mingwei SHENG ; Yiqi WENG ; Shusen WANG
Chinese Journal of Anesthesiology 2016;36(7):796-800
Objective To investigate the role of nuclear factor erythroid 2-related factor 2 (Nrf2) / heme oxygenase-1 (HO-1) signaling pathway in reduction of acute kidney injury following orthotopic liver transplantation (OLT) by hydrogen-rich saline in rats.Methods Thirty-two healthy adult male SpragueDawley rats,weighing 220-250 g,were randomly assigned into 4 groups (n =8 each) using a random number table:sham operation group (S group),OLT group,hydrogen-rich saline group (HS group),and all-trans retinoic acid (ATRA) group.Laparotomy was performed,and the related blood vessels were isolated in group S.The model of orthotopic autologous liver transplantation was established in OLT,HS and ATRA groups.Normal saline and hydrogen-rich saline 6 ml/kg were injected through the inferior vena cava at 5 min before the portal vein was clamped in OLT and HS groups,respectively.In group ATRA,Nrf2 inhibitor ATRA 7 mg/kg was injected intraperitoneally once a day for 2 consecutive days,the model of orthotopic autologous liver transplantation was established at 16 h after the last injection of ATRA,and the other treatments were similar to those previously described in group HS.At 6 h of reperfusion,blood samples were collected for determination of serum blood urea nitrogen (BUN),creatinine (Cr),interleukin10 (1L-10) and tumor necrosis factor-alpha (TNF-α) concentrations.After blood sampling,the lungs were removed for determination of malondialdehyde (MDA) content,superoxide dismutase (SOD) activity,expression of HO-1,Bcl-2 and Bax mRNA (by using real-time reverse transcriptase polymerase chain reaction),and HO-1 protein expression in lung tissues (by Western blot) and for microscopic examination.The damage to the renal tubules was scored.Results Compared with group S,the serum BUN,Cr and TNF-α concentrations were significantly increased,the serum IL-10 concentrations were decreased,the MDA content and renal tubular damage score were increased,the SOD activity was decreased,and the expression of HO-1 protein and mRNA,and Bcl-2 and Bax mRNA was up-regulated in group OLT (P< 0.05).Compared with group OLT,the serum BUN,Cr and TNF-α concentrations were significantly decreased,the serum IL-10 concentrations were increased,the MDA content and renal tubular damage score were decreased,the SOD activity was increased,the expression of HO-1 protein and mRNA and Bcl-2 mR-NA was up-regulated,and the expression of Bax mRNA was down-regulated in group HS (P<0.05).Compared with group HS,the serum BUN,Cr and TNF-α concentrations were significantly increased,the serum IL-10 concentrations were decreased,the MDA content and renal tubular damage score were increased,the SOD activity was decreased,the expression of HO-1 protein and mRNA and Bcl-2 mRNA was down-regulated,and the expression of Bax mRNA was up-regulated in group ATRA (P<0.05).Conclusion The mechanism by which hydrogen-rich saline reduces acute kidney injury following OLT is probably associated with activation of Nrf2/HO-1 signaling pathway in rats.