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ObjectiveTo study the mechanism of renal injury in urogenic sepsis and explore the effect of H2S on the expression of p38 mitogen-activated protein kinase (p38MAPK) and Cysteine protease 3(Caspase3) and apoptosis in renal tissue of urogenic sepsis. Hydrogen sulfide (H2S) has a wide range of biological effects and has a certain protective effect on the kidney. The main objective of this study is to investigate the effect of H2S on the expression of p38 mitogen-activated protein kinase (p38MAPK), cysteine proteinase 3 (Caspase3), and cell apoptosis in renal tissue of urogenic sepsis, and further to study the mechanism of urinary sepsis renal injury.MethodsNew Zealand rabbits (n=40) were divided into five groups Control, Sham, Sepsis, NaHS, and PAG, with eight rabbits in each group. The vital signs, blood routine white blood cell count, neutrophil count, and renal function (Cr, BUN) of five groups of New Zealand rabbits were recorded before the operation, 24 hrs after the operation, 48 hrs after the operation, and 72 hrs after the operation. 72 hrs after the operation, the left kidney tissue was taken for HE staining to observe the changes of cell morphology and structure of the kidney tissue. The apoptotic cells in renal tissue were labeled by Tunel assay (in situ terminal transferase labeling technique). The expression levels of p38MAPK and Caspase3 in renal tissue were tested by ELISA.Results The apoptosis indexes of renal tissue cells in group Control and group Sham were (5.65±2.43)% and (5.57±2.72)%, respectively, with no statistically significant difference (P>0.05). The apoptosis index of rabbit kidney tissue in group Sepsis was (25.26±2.70)%, which was significantly higher than that in group Control and group Sham (P0.05). Pairwise comparison between groups Sepsis, NaHS, and PAG showed statistically significant differences (P<0.05). The expression levels of group Sepsis and group PAG were significantly higher than that of group NaHS (P<0.05). The expression level of group PAG was significantly higher than that of group Sepsis (P<0.05).Conclusion H2S can alleviate renal damage caused by urine-derived sepsis, and its mechanism combined with H2S to suppress the expression of p38MAPK and Caspase3 in the renal tissue of urogenous sepsis, thereby reducing renal cell apoptosis Death.
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AIM:To explore the effect of hydrogen sulfide (H2S) on urosepsis-induced acute kidney injury.METHODS:New Zealand white rabbits were randomly divided into control group, sham group, model (sepsis) group, Na HS treatment (Na HS) group, and Na HS combined with TAK-242 (a TLR4 inhibitor) treatment (Na HS+TAK-242) group.After treatment for 72 h, HE staining was used to measure the histopathological changes of rabbit kidney.The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were detected by automatic biochemical analyzer.The serum levels of neutrophil gelatinase-associated lipocalin (NGAL) , kidney injury molecule 1 (KIM-1) , procalcitonin (PCT) , interleukin-1β (IL-1β) , interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by ELISA.The TLR4/MyD88/PI3K signaling pathway-related proteins in the kidney were determined by Western blot.RESULTS:Compared with control group, obvious damage was observed in the kidneys of septic rabbits, but the kidneys were markedly improved by treatment with Na HS.The levels of BUN, SCr, NGAL, KIM-1, PCT, IL-1β, IL-6 and TNF-αin the septic rabbits were higher than those in control group, and decreased significantly in Na HS group and Na HS+TAK-242 group.The protein levels of TLR4, MyD88, p-PI3K and p-Akt in septic rabbit kidneys were higher than those in control group.However, Na HS or Na HS+TAK-242 inhibited the activation of TLR4/MyD88/PI3K signaling pathway in the kidneys of septic rabbits.CONCLUSION:H2S play a protective effect on the rabbits with urosepsis-induced acute kidney injury by blocking TLR4/MyD88/PI3K signaling pathway to inhibit inflammatory response.
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<p><b>OBJECTIVE</b>To construct a replication-incompetent recombinant adenovirus mediating short hairpin RNA (shRNA)-induced tissue factor gene silencing in the islet.</p><p><b>METHODS</b>Four pairs of complementary oligonucleotides were designed and synthesized to create double-stranded oligonucleotides (ds oligo). The ds oligos were cloned into Pentr/U6 vector to construct the shuttle plasmid pENTR/U6-shRNA, which was transduced into human islets via liposome after sequence verification. The plasmid with the best silencing effect was identified by real-time RT-PCR, followed by homologous recombination with the adenovirus backbone plasmid. The functional clone was transfected into 293A cells to amplify the adenovirus, whose silencing effect against TF expression was tested using real-time RT-PCR and Western blotting.</p><p><b>RESULTS</b>The pENTR/U6-shRNA shuttle plasmid was constructed and verified by sequencing. The recombinant adenovirus-mediated shRNA against TF was constructed, and real-time RT-PCR and Western blotting demonstrated that the strongest silencing effect of the adenovirus against TF occurred on the 4th day following islet transfection.</p><p><b>CONCLUSION</b>Replication-incompetent recombinant adenovirus-mediated shRNA against TF has been successfully constructed, which has good silencing effect against TF expression in human islet in vitro.</p>