ABSTRACT
Objective:To screen out the potential key genes of sepsis-associated acute kidney injury (AKI), and provide theoretical and experimental evidence for the treatment of sepsis-associated AKI.Methods:① Bioinformatics analysis: two gene expression datasets (GSE30718 and GSE53773) were downloaded for bioinformatics analysis from the Gene Expression Omnibus (GEO). These two datasets recorded mRNA microarray data from kidney biopsies before and after kidney transplantation, and a subset of patients developed AKI after kidney transplantation. Differential analysis was conducted, and the genes with the same differential expression and a higher area under the receiver operator characteristic curve (AUC) in both databases were used as the target gene for subsequent cell experiments. ② Cell validation experiment: human proximal renal tubular cells HK2 were cultured in vitro, and lipopolysaccharide (LPS) was used for establishing LPS-HK2 cell model (LPS 10 mg/L for 6 hours, LPS model group), and the blank control group was set. Then, small interfering RNA (siRNA) technology was used to knock down the target gene obtained by bioinformatics analysis in LPS-HK2 cells (gene knockdown group), and a gene negative control group was set. The real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-qPCR) technique was used to detect the expression of the target gene in HK2 cells. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors in the cell supernatants. Western blotting was used to detect the expressions of key apoptosis proteins. Results:① Results of bioinformatics analysis: 325 genes in the two datasets showed the same expression trend, of which 144 were significantly down-regulated and 181 were significantly up-regulated, while the expression difference of secretory leukocyte protease inhibitor (SLPI) in the two datasets was both statistically significant. Further receiver operator characteristic curve (ROC curve) analysis confirmed that the SLPI expression in GSE30718 and GSE53773 datasets had a high diagnostic efficiency for AKI, with AUC of 0.83 and 0.92, respectively. Therefore, SLPI was selected as the target gene for subsequent cell validation experiment. ② Cell validation experiment: the RT-qPCR analysis showed that the expression of SLPI in LPS-HK2 cells of the LPS model group was significantly higher than that of the blank control group (2 -ΔΔCT: 1.80±0.14 vs. 1.00±0.11, P < 0.01), and the change trend was the same with the results of bioinformatics analysis. Furthermore, knockdown SLPI gene analysis showed that the levels of inflammatory factors in LPS-HK2 cells supernatants in the gene knockdown group were significantly higher than those in the negative control group [Interleukin-6 (IL-6, ng/L): 509.58±27.08 vs. 253.87±75.83, IL-1β (ng/L): 490.99±49.52 vs. 239.67±26.97, tumor necrosis factor-α (TNF-α, ng/L): 755.22±48.66 vs. 502.06±10.92, all P < 0.01]. The above results indicated that SLPI could inhibit the inflammatory response of HK2 cells induced by LPS. The expressions of key apoptosis proteins Bax and caspase-3 in LPS-HK2 cells in the gene knockdown group were significantly higher than those in the negative control group [Bax protein (Bax/GAPDH): 1.38±0.12 vs. 1.00±0.10, caspase-3 protein (caspase-3/GAPDH): 1.44±0.15 vs. 1.00±0.11, both P < 0.05], and Bcl-2 expression was significantly decreased (Bcl-2/GAPDH: 0.83±0.08 vs. 1.00±0.05, P < 0.05), the above results indicated that SLPI could inhibit the apoptosis of cells in the inflammatory response. Conclusion:SLPI can inhibit the inflammatory response and apoptosis of HK2 cells induced by LPS, which may be involved in the protective mechanism of renal tubular cells in the response to sepsis, and is a potential target for the treatment of sepsis-associated AKI.
ABSTRACT
ObjectiveTo analyze the related data of patients with severe pesticide poisoning in Tongling area, put forward the countermeasures,and provide available basis to improve the level of prevention and treatment of the disease.Methods Forty patients with severe pesticide poisoning admitted to the Department of Critical Care Medicine of Tongling People's Hospital Affiliated to Wannan Medical College from August 2014 to November 2015 were analyzed retrospectively, the clinical data including distributions of sex, ages, kinds of poisoning drugs, causes of poisoning, time from intoxication to admission into a hospital, rescue therapy, prognosis etc.Results In the 40 patients, there were 12 males and 28 females, the ratio between male to female was 1∶2.33, and the number of women was significantly higher than that of men. Their ages ranged from 40 - 60 years, accounting for 37.5% (15/40). The toxic agent the most commonly seen was organic phosphorus pesticide (15 cases, accounting for 37.5%), and cases with mixed poisoning by taking two or more than two kinds of toxic material accounted for 25.0% (10/40). In addition, the cause of poisoning was mainly due to family quarrels (33 cases, accounting for 82.5%). In regard of time between taking toxic material to admission into a hospital, only did 3 cases admit into the hospital within 1 hour (7.5%), and the majority was within 3 hours (15 cases, accounting for 37.5%). The comprehensive treatments were as follows: clear means to clear the toxin,solution (to use antidote to detoxify), maintainance (to maintain the basic vital signs) and exclusion (to exclude other complications early); the curative effect of comprehensive treatments was satisfied, and 3 cases died, accounding for 7.5%.Conclusions The majority of patients with severe pesticide poisoning in Tongling city are adult females, the main cause of taking toxic material is due to family quarrels, and the toxic agent the most commonly seen is organic phosphorus pesticide. Directing to the above high risk factors, the prevention and treatment are carried out, anticipating the incidence, mortality and medical cost can be reduced significantly.