Effects of Ling Qi Juan Gan capsule drug-containing serum on PDGF-induced proliferation and JAK/STAT signaling of HSC-T6 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To analyze the effects of Ling Qi Juan Gan capsule drug-containing serum at different concentrations on the platelet-derived growth factor (PDGF)-induced proliferative capabilities of and JAK2 and p-STAT3 protein expression in hepatic stellate cells (HSC) using an in vitro system.</p><p><b>METHODS</b>Twenty-five Sprague-Dawley rats were randomly divided into five equal groups for intragastric administration of physiological saline (10 ml/kg; group A), Fufang Biejia Ruangan tablet solution (1.5 g/kg; group B), or Ling Qi Juan Gan capsule solution at low dose (2.125 g/kg; group C1), mid dose (4.25 g/kg; group C2), or high dose (8.5 g/kg; group C3). The post-administration serum isolated from each rat (200 ml/L) was used to treat the HSC-T6 cell line following induction by PDGF (10 ng/ml). At 24, 48 and 72 h post-exposure, the cells' proliferation was measured using the Cell Counting Kit-8 (CCK-8) colorimetric assay. In addition, at 24 h post-exposure the expression of JAK2 and p-STAT3 was measured by western blotting (expressed as grey scale intensity). Multiple group comparison of repeated measures data was made by one-way ANOVA with Student-Newman-Keuls post hoc test.</p><p><b>RESULTS</b>Compared to group A, groups C2 and C3 had significantly higher inhibited proliferation at all post-exposure time points examined (24 h: A = 1.550 +/- 0.065, C2 = 1.335 +/- 0.106, C3 = 1.241 +/- 0.205; 48 h: A = 1.311 +/- 0.650, C2 = 1.090 +/- 0.106, C3 = 0.909 +/- 0.191; 72 h: A = 1.039 +/- 0.103, C2 = 0.719 +/- 0.116, C3 = 0.641 +/- 0.110, F = 36.292, all P less than 0.05); in contrast, compared to group A, group C1 showed no inhibition of proliferation at 24 h (1.522 +/- 0.128, P = 0.717) but showed significantly higher inhibition of proliferation at 48 h and 72 h (1.153 +/- 0.183 and 0.753 +/- 0.210, respectively, F = 36.292, P less than 0.05). Compared with group A, all Ling Qi Juan Gan capsule-containing serum-treated groups showed significantly lower expression of both JAK2 (A = 1.605 +/- 0.024 vs. C1 = 1.170 +/- 0.042, C2 = 0.842 +/- 0.036, C3 = 0.555 +/- 0.036, F = 43.091) and p-STAT3 (A = 1.401 +/- 0.030 vs. C1 = 1.229 +/- 0.025, C2 = 0.668 +/- 0.034, C3 = 0.630 +/- 0.026, F = 78.426, all P less than 0.01).</p><p><b>CONCLUSION</b>Ling Qi Juan Gan capsule drug-containing serum can inhibit the proliferation of HSC-T6 cells in a dose-dependent manner and cause an overall decrease in the expression of JAK2 and p-STAT3 in activated HSC, thereby leading to a suppression of the JAK/STAT signal transduction pathway.</p>

Role of serum from asphyxiated neonates in the inducement of human renal tubular cell adhesion to neutrophils / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the role of serum from asphyxiated neonates in the inducement of human renal proximal tubular epithelial cells (HK-2) adhesion to neutrophils and possible mechanisms.</p><p><b>METHODS</b>HK-2 cells were cultured randomly with 20% serum from neonates (1, 3, and 7 days after asphyxia), pyrrolidine dithiocarbamate (PDTC) or placebo. The activity of myeloperoxidase (MPO), an indicator of adhesion ability of HK-2 cells to neutrophils in suspensions, was detected by the biochemistry assay. Intercellular adhesion molecule-1 (ICAM-1) and nuclear factor-kappaB (NF-kappaB) of HK-2 cells were examined with the immunohistochemical staining.</p><p><b>RESULTS</b>The expression of MPO in the post-asphyxial 1-day serum treatment group were significantly higher than that in the PDTC treatment and the control groups as well as the post-asphyxial 3 and 7-day serum treatment groups (P<0.01). The expression of ICAM-1 and NF-kappaB in the post-asphyxial 1-day serum treatment group was also significantly higher than that in the other groups (P<0.01).</p><p><b>CONCLUSIONS</b>Serum from asphyxiated neonates can induce HK-2 cell adhesion to neutrophils, possibly through activating NF-kappaB and increasing the synthesis and expression of ICAM-1 on the surface of renal tubular epithelial cells.</p>

Role and mechanism of Salvia miltiorrhiza Bunge in alleviating injury induced by hypoxia/reoxygenation in HK-2 cells / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Salvia miltorrhiza Bunge (SMB) is a traditional Chinese herb, which is considered to promote blood flow and remove blood stasis. This study examined whether SMB can alleviate injury induced by hypoxia/reoxygenation (H/R) in human kidney proximal tubular cells-2 (HK-2 cells).</p><p><b>METHODS</b>There were 3 experimental groups: control, H/R injury and SMB-treated H/R injury. H/R injury of HK-2 cells was induced by first covering the cells with and then removing liquid paraffin wax. Different concentrations of compound SMB solution (0.05%, 0.10%, 0.15% or 0.20%) were administered to the SMB-treated H/R injury group before the hypoxic injury. After 4, 12 and 24 hrs of hypoxia and 4, 12, 24 and 48 hrs of reoxygenation, morphologic changes of HK-2 cells were observed under an inverted microscope. Cell viability was measured by the MTT method. Lactate dehydrogenase (LDH) activity in the culture supernatants was assayed using biochemical methods; TNF-alpha levels were determined by radioimmunoassay (RIA).</p><p><b>RESULTS</b>The number of HK-2 cells was significantly reduced in the H/R injury group after hypoxia, and reached a nadir 24 hrs after hypoxia treatment. Various concentrations of SMB-treated groups showed significantly greater number of HK-2 cells than the H/R injury group. SMB solution (0.10%) produced the best effect. The levels of LDH and TNF-alpha in the H/R injury group were significantly increased, and reached a peak between 24 hrs of hypoxia and 4 hrs of reoxygenation when compared to the control group. Pre-treating with 0.10% SMB resulted in significantly lower levels of LDH and TNF-alpha than in the untreated H/R injury group at various time points of H/R.</p><p><b>CONCLUSIONS</b>SMB has protective effects against H/R injury of HK-2 cells, possibly through inhibition of inflammatory cytokines.</p>

Effect of Erythropoietin on Apoptosis of Human Renal Tubular Cells Induced by Postasphyxial-Serum of Neonates / 实用儿科临床杂志

Objective To explore the effect of erythropoietin(EPO) on apoptosis of human renal tubular(HK-2) cells induced by postasphyxial-serum of neonates.Methods HK-2 cells were used as target cells.The experiment was divided into 4 groups,control group(n=8):HK-2 cells were maintained in standard medium;asphyxia group(n=8):HK-2 cells were treated with serum obtained from neonates with asphyxia.Each culture medium replaced with 200 mL/L suffocation DMEM/F12 serum culture medium;EPO pretreatment group(n=8):HK-2 cells were pretreated 24 h with serum contains 5?104 IU/L EPO,and then deal as asphyxia group;EPO and 5-hydroxydecanoic acid sodium salt(5-HD) pretreatment group(n=8): HK-2 cells were pretreated 24 h with serum contains 5?104 IU/L EPO and 500 ?mol/L 5-HD,and then deal as asphyxia group.All cells were cultured at 37 ℃ in humidified atmosphere with 50 mL/L CO2 for 24 h.The apoptosis rate of HK-2 cells was detected by flow cytometer.The expressions of Caspase-3 and X-linked inlnibitor of apoptosis protein(XIAP) of HK-2 cells were detected by using immunohistochemical method.Results Compared with control group,after stimulated with postasphyxial-serum,the apoptosis rate and the expression of Caspase-3 of HK-2 cells were significantly increased(Pa

Protective Effects of Astragalus on Human Renal Tubular Cells Injury Induced by Postasphyxial-Serum from Neonate / 实用儿科临床杂志

Objective To explore the protective effects of astragalus on injury of renal tubular cells(HK-2) induced by postasphyxial-serum from neonate.Methods Human renal proximal tubular cell line HK-2 cells were taken as subjects.The experiment was designed as:control group,model group,astragalus pretreatment group.The astragalus pretreatment group had 5 subgroups,which were pretreated for 24 hours.The serum of neonates who suffured asphyxia for 1 day,which concentration were 200 mL/L(volume fraction),were applied as attacking factor.The injury of morphology were observed under inverted microscope.The cell viability was measured by methyl thiazolyl tetrazolium methods.The leakage rate of lactate dehydrogenase(LDH) was determined by biochemical methods.Results Compared with control group,cell morphous changed,leakage rate of LDH elvated and cell survival rate decreased in model group,the variances were obviously significant(Pa

Role of Na~+/H~+ Exchanger 1 on Injury of Human Renal Tubular Cell Induced by Postasphyxial-Serum of Neonates / 实用儿科临床杂志

Objective To explore the role of Na~+/H~+ exchanger 1(NHE1) in injury of human renal tubular cells(HK-2) induced by postasphyxial-serum of neonates.Methods HK-2 was used as the target cell.The attacking concentration of postasphyxial-serum of neonates was 200 mL/L.First,the experiment was designed as control group and asphyxia group,the expression of NHE1 in the HK-2 was detected by immunohisto chemical method in the cells.Second,the experiment group was designed as control group,asphyxia group,and pretreatment with 5-N-Ethyl-N-isopropylamiloride(EIPA) group,then the change of morphology was observed under inverted microscope,and the cell viability was measured by methyl thiazolyl tetrazolium(MTT) method,and the leakage rate of lactate dehydrogenase(LDH) was determined by biochemical methods.Results Compared with blank control group,the expression of NHE1 in the HK-2 was increased signi-ficantly in asphyxia group,the changes of morphology of HK-2 was most serious and obvious,the cell viability decreased,and the leakage rate of LDH increased significantly in asphyxia group.But compared with asphyxia group,the change of morphology of HK-2 was greatly improved,the cell injury was decreased obviously,the leakage rate of LDH was increased and viability was decreased in pretreatment group in a dose 2 ?mol/L.Conclusions The postasphyxial-serum may induce the expression of NHE1,which plays an important role in injury of renal tubular cell induced by postasphyxial-serum in neonates,and inhibiting activity of NHE1 may relieve the injury of renal tubular cells induced by postasphyxial-serum in neonates.

Effect of Postasphyxial-Serum of Neonate in Inducing Injury of Human Renal Tubular Cell / 实用儿科临床杂志

Objective To investigate the role of postasphyxial-serum of neonate in inducing injury of human renal proximal tubular cells(HK-2 cells).Methods HK-2 cells were used as target cell.The neonatal different concentration postasphyxial-serum of 1,3,7 days after asphyxia were used as attacking means.The experimental groups were divided into 15 groups:the 2.5%,5.0%,10.0%,(20.0%) attacking concertration groups of 1,3,7 day after asphyxia and control group of each concertration.The culture medium and concertration of the control group and the experimental groups were the same.The changes of morphology were observed under inverted microscope,the cell viability was measured by 3-(4,5-dimethyl-2-thiazoly1)-2,5-diphenyl-2H-tetrazolium bromide(MTT) method and the leakage rate of lactate dehydrogenase(LDH) was determined by biochemical methods.Results Compared with control group,the changes in morphology of HK-2 were most serious and obvious,the cell viability were obviously decreased(all P

Effect of Postasphyxial-Serum in Neonate Inducing Apoptosis of Renal Tubular Cells / 实用儿科临床杂志

Objective To investigate the role and intracellular signal transduction mechanism in the injury of renal tubular cells induced by postasphyxial-serum in neonate.Methods Human renal proximal tubular cell(HK-2 cell) was used as target cell. The experiment was designed as:control group, asphyxia group ,and pyrrolidine dithiocarbamate (PDTC)blocking group. The attacking concentration of serum was 20%, and the apoptosis rate of HK-2 cells was detected by flow cytometer.Results Compared with controls[(13.3?1.70)%],after being stimulated with postasphyxial-serum, the apoptosis rate of HK-2 cells of asphyxia group [(46.73?3.68)%] and PDTC blocking group [(31.19?2.79)%]were significantly increased(P

Influence of Erythropoietin on Signal Conduction of Injury of Human Renal Tubular Cell Induced by Postas-phyxial-Serum of Neonate / 实用儿科临床杂志

Objective To explore the influence of erythropoietin(EPO)on signal conduction mechanism of injury of renal tubular cells(HK-2) induced by postasphyxial-serum of neonates.Methods Heavy term asphyxia neonate were selected which amounted to 50 cases(28 male and 22 female)who had no obvious infectious diseases and immune suppression preparation,and their family consented to drawing their venous blood 5 mL after asphyxia 1 day,and put on centrifuge for 10 minutes with 2 500 r/min,then in aqueous bath for 30 minutes to inactivate complement at 56 ℃,and undertook filtration sterilization by micropore filter.The attacking concentration of serum was 200 mL/L which was prepared by adopting Dulbecco′s Modified Eagle Medium/Nutrient Mixture F12 culture fluid.HK-2 was used as the target cel1.Before attack,the experiment was designed as:blank control group,group of pretreatment with EPO(5?104 IU/L EPO medium preconditioned for 24 hours).After attack,at the 15 minutes,1 hour,2 hours,experiment was designed as:attacking control group and attacking group preconditioned with EPO at each time point.The nuclear translocation of nuclear factor ?B(NF-?B) was detected using indirect immunofluorescence.The amount of I-?B? change by NF-?B restraining was detected by Western blot,the results were scanned into computer,and image analysis software of Image-Pro Plus was used to analyze the strap.The strap integral optical density value represented the quantity of protein.Results Before attack,compared with bland control group,the nuclear translocation of NF-?B increased and amount of I-?B? decreased in group of pretreatment with EPO(Pa

Protective Effects of Diazoxide on Injury of Human Renal Tubular Cell Induced by Serum Obtained from Neonates with Asphyxia / 实用儿科临床杂志

Objective To explore the protective effects of diazoxide on injury of human renal tubular cell(HK-2)induced by serum obtained from neonates with asphyxia.Methods HK-2 cells was used as the target cel1.The attacking concentration of serum obtained from neonates with asphyxia was 200 mL/L.The experiment was designed as 3 groups.HK-2 cells were divided into control group,asphyxia group,and diazoxide group.Control group:joined nutrient fluid including 100 mL/L embryo cow blood serum.Asphyxia group:joined nutrient fluid including the isometric 200 mL/L serum obtained from neonates with asphyxia.Diazoxide group:the diazoxide was joined nutrient including the isometric 200 mL/L serum obtained from neonates with asphyxia fluid.The diazoxide density finally was 100 ?mol/L.Then the change of morphology was observed and photographed under inverted microscope,and the cell viability was measured by methyl thiazolyl tetrazolium method,and the leakage rate oflactate dehydrogenase(LDH)was determined by biochemical methods.Results Under inverted microscopy,HK-2 cells in control group pastes the wall to be good,assumes the paving stone type,into flat polygon,fission many,the cell arrangement was close,connection large expanse,quantity were many.Compared with control group,the HK-2 cell to suffer injury obviously,the shape changed,become the anomalous circular or the ellipse by the model flat polygonal cell,the intercellular space crevice enlarged,the connection was loose,intercellular space obviously many cell fragmented.Living cell quantity reduced obviously,the cell vigor dropped,and the leakage rate of LDH increased significantly in asphyxia group(P

Effect of Postasphyxial-Serum in Neonate on Expression of Omi/HtrA2 in Renal Tubular Cells / 实用儿科临床杂志

Objective To explore the effect of postasphyxial-serum in neonate on expression of serine protease Omi/HtrA2 in renal tubular cells(HK-2).Methods Human renal proximal tubular cell line HK-2 cell was used as target cell.The cultural cells in orifice were divided into control group and asphyxia-serum attacking group.Blood was cowected from asphyxia newborns by means of femoral venous puncture,then the serum was garthered,anticoagulated by liquemie,3 000 r/min centrifuged 20 min,abstracted serum,thermostatic waterbathed the serum at 56 ℃,so that to inactivate addiment,filtered germ by micropore filte,the attacking concentrtion of serum was 200 mL/L,the cells of the asphyxia-serum attacking group were attacked by asphyxia-serum,and the cells of control group were cultivated with normal nutritive medium when the cells was needed.After 24 hours,the cells were tixed,then the expression of Omi/HtrA2 in cytoplast was detected by the use of immunohistochemical method.Results Omi/HtrA2 was inaurate or yellow brown and localized to the cytoplast.The rate of the cell expressed Omi/HtrA2 was(9.0?2.5)% in control group,after stimulated with postasphyxial-serum,in asphyxia group the rate of the cell expressed Omi/HtrA2 was(25.15?3.5)%,there was significant difference between 2 groups(t=-15.322 P

Role of Erythropoietin in Relieving Injury of Human Renal Tubular Cell Induced by Postasphyxial-Serum of Neonates / 实用儿科临床杂志

Objective To investigate the role of erythropoietin(EPO)in relieving the injury of human renal tubular cells (HK-2) induced by postasphyxial-serum of neonates.Methods Human renal proximal tubular cell(HK-2) was used as the target cell.The experiment was designed as control group, asphyxia group,and group of pretreatment with EPO. The attacking concentration of serum was 200 mL/L,then the changes of morphology were observed under inverted microscope,and the cell viability was measured by 3-(4,5-dimethy lthiazcl-2-yl)-2,5-diphenyl-tetazolium bromide(MTT) methods,and the leakage rate of lactate dehydrogenase(LDH) was determined by biochemical methods.Results Compared with control group,the change in morphology of HK-2 was most serious and obvious,and the leakage rate of LDH increased significantly,and the cell viability decreased obviously in asphyxia group.But compared with asphyxia group,the change in morphology of HK-2 was obviously improved,and the leakage rate of LDH decreased and the cell viability increased in group of pretreatment with EPO in a dose-dependent manner except the group of 1 IU/mL.Conclusion EPO can play the role in relieving the injury of renal tubular cells induced by postasphyxial-serum in neonates.

Expression of Hypoxia Inducible Factor-1? in Human Renal Proximal Tubular Cells Injuried by Hypoxia/Reoxygenation and the Intervention Effect of Danshen / 实用儿科临床杂志

Objective To explore expression of hypoxia inducible factor-1?(HIF-1?)in kidney cells during hypoxia/reoxygenation injury,and to study the effect of Danshen on prevention of the hypoxia/reoxygenation injury to cells.Methods Human renal proximal tubular cells(HK-2)were used as the target cell.There were 3 groups:control group,model group,Danshen group.hypoxia/reoxygenation models after neonatal asphyxia were established with liquid paraffin covering method.Expression of HIF-1? were detected with strcp avidin biotin complex(SABC)immunohistochemistry at following different time points:hypoxia 1,4,8,12,24 h,which means 1,4,8,12,24 h respectively after hypoxia;and hypoxia/reoxygenation 1,4,8,12,24 h,which means 1,4,8,12,24 h respectively after hypoxia/reoxygenation.Results HIF-1? was expressed mainly in HK-2's nucleus.There had low expression of HIF-1? in HK-2 cells under the normal culture,and its expression level kept rising quickly during hypoxic/reoxygenatic culture,until 4 h after the beginning of reoxygenation.Compared with the study group,the expression level of HIF-1? in HK-2 cells of the Danshen group were significantly lower at different time points(Pa

Effect of Postasphyxial-Serum in Neonate on the Expressions of Bcl-2-Antagonist of Cell Death and Bcl-2-Associated X Protein in Renal Tubular Cells / 实用儿科临床杂志

Objective To explore the effect of postasphyxial-serum in neonate on the expressions of Bcl-2-antagonist of cell death(BAD)and Bcl-2-associated X protein(BAX)in renal tubular cells(HK-2).Methods HK-2 cells were used as target cells.The experiment were divided into control group,asphyxia group and pyrrolodine dithiocarbamate(PDTC)blocking group.Control group:DMEM culture fluid was not contained asphyxia blood serum in every group;asphxia group:DMEM culture fluid contained 20 mL/L asphyxia blood serum in every group;PDTC blocking group:DMEM culture fluid contained 20 mL/L asphyxia blood serum and 40 ?mol/L PDTC in every group.The expressions of both BAD and BAX on cytoplast were detected by immunohistochemical method.Results Calculated Points according to HSCORE,compared with controls group[(1.97?0.26)and(1.77?0.11)],after stimulated with postasphyxial-serum,the expressions of both BAD and BAX of HK-2 cells of asphyxia group[(2.73?0.20)and(2.44?0.13)] and PDTC blocking group[(2.38?0.13)and(2.17?0.08)] significantly increased[F(BAD)=28.61,F(BAX)=15.51 Pa