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@#[摘 要] 目的：探讨miR-502-3p通过靶向GTP结合蛋白2（GTPBP2）调控结直肠癌干细胞（CCSC）增殖、细胞周期和凋亡的分子机制。方法：利用免疫磁珠分选技术从结直肠癌细胞HCT116中分选CCSC（CD133+CD44+双阳性细胞和CD133-CD44-双阴性细胞），用qPCR法检测细胞中miR-502-3p表达水平。利用脂质体转染法分别将miR-NC、miR-502-3p、si-miR-NC、si-miR-502-3p、miR-502-3p+vector和miR-502-3p+GTPBP2转染至CD133+CD44+细胞中，记作miR-NC、miR-502-3p、si-miR-NC、si-miR-502-3p、miR-502-3p+vector和miR-502-3p+GTPBP2组。用qPCR法检测细胞中miR-502-3p、GTPBP2 mRNA表达水平，MTT法、流式细胞术分别检测细胞增殖率、细胞周期和凋亡率，WB法检测细胞中Ki67、CDK1、Bcl2、BAX和GTPBP2蛋白的表达水平。双荧光素酶报告基因实验验证miR-502-3p与GTPBP2基因的靶向关系。结果：CD133+CD44+细胞中miR-502-3p表达水平显著低于CD133-CD44-细胞（P<0.01）。与miR-NC组比较，miR-502-3p组细胞增殖率、S期细胞比例显著降低（均P<0.01），凋亡率、G0/G1期细胞比例显著升高（均P<0.01），细胞中Ki67、CDK1、Bcl2蛋白表达均显著下调（均P<0.01）、BAX蛋白表达显著上调（P<0.01）。miR-502-3p靶向调控GTPBP2的表达，过表达GTPBP2可逆转上调miR-502-3p对CCSC增殖、周期和凋亡的作用。结论：上调miR-502-3p表达抑制CCSC增殖和阻滞细胞周期、诱导凋亡，其作用机制可能与过表达GTPBP2有关。
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Objective::To explore the pharmacological mechanism of Xiao Xianxiongtang in treating type 2 diabetes mellitus (T2DM) by network pharmacology. Method::The main active ingredients, corresponding targets and target genes of Xiao Xianxiongtang were searched on Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) website. Relevant target genes of T2DM were obtained through Gene Cards. The targets of drug active ingredients were mapped to the targets of T2DM, and the intersection targets were obtained as the predictive targets of Xiao Xianxiongtang on T2DM. Cytoscape 3.7.1 software was used to construct the drug active ingredient-intersection target network model and select the key active ingredients. Interactive protein-protein interaction network (PPI) was constructed by STRING website, and key target genes were selected. Gene function analysis (GO) and enrichment analysis based on the Kyoto encyclopedia of genes and genomes (KEGG) pathway were performed on the intersecting targets using DAVID6.8 online tool. Result::Xiao Xianxiongtang had 30 active ingredients, 156 relevant targets, 14 key active ingredients and 18 key target genes on T2DM. GO analysis showed that the biological functions of Xiao Xianxiongtang in the treatment of potential genes of T2DM mainly involved transcriptional regulation, oxidative stress, protein binding and inflammatory reaction. KEGG pathway enrichment showed that the main pathways of Xiao Xianxiongtang in the treatment of T2DM were hypoxia inducible factor-1 (HIF-1) signaling pathway, tumor necrosis factor (TNF) signaling pathway, Toll-like receptor signaling pathway and thyroid hormone signaling pathway, phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling pathway, hepatitis B, hepatitis C, tyrosine kinase receptor2(ErbB) signaling pathway, calcium signaling pathway and nuclear factor-kappa B (NF-κB) signaling pathway. Conclusion: Xiao Xianxiongtang is a multi-component, multi-target and multi-pathway process in the treatment of T2DM. It plays an important role in the treatment of T2DM by regulating transcription, oxidative stress, protein binding and inflammatory reaction. Conclusion::The mechanism of Xiao Xianxiongtang in treating T2DM may alleviate insulin resistance, increase insulin sensitivity and reduce blood sugar by inhibiting the secretion of inflammatory factors, participating in anti-inflammatory response, reducing oxidative stress, increasing intracellular calcium concentration, blocking glucagon signaling pathway and activating PI3K/Akt pathway.
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Objective: To explore the mechanism of Dahuang Huanglian Xiexintang in the treatment of type 2 diabetes mellitus based on network pharmacology. Method: Major chemical constituents, corresponding targets and target genes of Dahuang Huanglian Xiexintang were obtained by Traditional Chinese Medicine Systems Pharmacology(TCMSP), and target genes of type 2 diabetes mellitus were obtained by GeneCards. The target genes of drug and disease were mapped to predict target genes of Dahuang Huanglian Xiexintang for type 2 diabetes mellitus. Cytoscape3.7.1 software was used to construct the compound-target network and protein-protein interaction network (PPI) of traditional Chinese medicine. Gene ontology (GO) analysis of potential genes and enrichment analysis of gene encyclopedia kyoto encyclopedia of genes and genomes (KEGG) pathway were carried out using DAVID 6.8 online tool. Result: There were 17 active ingredients, 94 related targets, 17 key active ingredients and 16 key targets in Dahuang Huanglian Xiexintang on type 2 diabetes mellitus. GO analysis showed that the biological functions of potential genes of Dahuang Huanglian Xiexintang in the treatment of type 2 diabetes were mainly related to oxidative stress, apoptosis, protein binding, inflammatory reaction, et al. KEGG pathway enrichment results showed that the pathways of potential genes of Dahuang Huanglian Xiexintang in the treatment of type 2 diabetes mainly involved hypoxia inducible factor(HIF), tumor necrosis factor(TNF), phosphatidylinositol 3 kinase/protein kinase B(PI3K/Akt), nuclear transcription factor-кB(NF-кB), and vascular endothelial growth factor(VEGF) signaling pathways. Conclusion: Dahuang Huanglian Xiexintang is a complex process of multi-component, multi-target and multi-pathway in the treatment of type 2 diabetes mellitus. It plays an important role in the treatment of type 2 diabetes mellitus by participating in oxidative stress, apoptosis, protein binding and inflammatory reaction.
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Objective To investigate the effects of pyrrolidine dithiocarbamate (PDTC) on oxidative stress and mitochondrial function of lung tissue in mice with acute lung injury (ALI) induced by lipopolysaccharide (LPS). Methods Forty female Balb/c mice were randomly divided into normal saline (NS) control group, LPS model group, PDTC group, and PDTC+LPS group, with 10 mice in each group. The model of mice with ALI was reproduced by intraperitoneal injection of 15 mg/kg LPS. PDTC was administered intraperitoneally with 50 mg/kg PDTC 1 hour before LPS treatment in the PDTC+LPS group. The mice in NS control group was given intraperitoneal injection of 0.1 mL NS only, and those in PDTC group was given intraperitoneal injection of 50 mg/kg PDTC only. The mice were sacrificed at 24 hours after model reproduction, and the lung tissues were harvested. The total antioxidant capacity (T-AOC) of lung tissue was measured by spectrophotometric kits. The content of malondialdehyde (MDA) was determined by thiobarbituric acid reactive substances assay. The protein expressions of superoxide dismutases (SOD1, SOD2) and catalase (CAT) in lung tissue were determined by Western Blot. Mitochondria from mouse lungs were isolated, and adenosine triphosphate (ATP) synthesis was measured with a luciferase/luciferin-based approach. The mitochondrial membrane potential (ΔΨm) was estimated by using Rhodamine. The mRNA expressions of mitochondrial uncoupling proteins (UCPs) were determined by reverse transcription-polymerase chain reaction (RT-PCR). Results LPS stimulation could significantly increase oxidative stress in lung tissue of mice and lead to mitochondrial dysfunction. The results showed that the protein expressions of T-AOC and SOD1 were decreased, the level of MDA was increased, the ATP synthesis was decreased in the mitochondrial, the ΔΨm was decreased, and the mRNA expression of UCP2 was decreased. However, there was no significant change in the expressions of SOD2, CAT in lung tissue and UCP1, UCP3 in the mitochondria. Pretreatment with PDTC could obviously alleviate the increase in LPS-induced oxidative stress in lung tissue and mitigate mitochondrial dysfunction. Compared with the LPS model group, T-AOC in lung tissue of PDTC+LPS group was significantly increased (U/g: 0.35±0.08 vs. 0.31±0.07), the level of MDA was significantly decreased (μmol/mg: 13.29±1.13 vs. 17.54±1.72), the protein expression of SOD1 was significantly upregulated (SOD1 protein:1.13±0.11 vs. 0.71±0.09), ATP synthesis was significantly increased in the mitochondrial (μmol/mg: 49.23±5.42 vs. 36.92±2.21), ΔΨm was significantly increased (mV: 226.03±11.69 vs. 194.86±7.79), and the mRNA expression of UCP2 was significantly increased (2-ΔΔCt: 0.88±0.06 vs. 0.73±0.04). The differences were statistically significant (all P < 0.05). In lung tissue of normal mice, PDTC treatment also had the effect of anti-oxidizing, reducing oxidative stress and promoting ATP synthesis in the mitochondrial. Compared with the NS control group, the level of T-AOC (U/g: 0.49±0.09 vs. 0.43±0.06) and the protein expressions of SOD2 and CAT (SOD2 protein: 1.33±0.08 vs. 1.00±0.11, CAT protein: 1.39±0.08 vs. 1.00±0.11), and ATP synthesis in the mitochondrial of PDTC group was significantly increased (μmol/mg: 61.53±4.92 vs. 53.33±3.20), MDA was significantly decreased (μmol/mg:10.27±1.25 vs. 12.27±1.36), with statistical differences, but had no effect on the protein expression of SOD1 in lung tissue and ΔΨm and UCPs mRNA expressions in mitochondrion. Conclusions LPS can induce ALI in mice, increased oxidative stress in lung tissue, and induce mitochondrial dysfunction by inhibiting ATP synthesis. PDTC pretreatment has anti-oxidative effect on LPS-induced ALI, and can mitigate mitochondrial dysfunction.
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Objective To compare the impact of mean lactate concentration and lactate variability on postoperative outcome after cardiac surgery and non-cardiac surgery in critical patients, and to explore the prognostic value of the first lactate and the highest lactate during the first 24 hours in intensive care unit (ICU). Methods A retrospective study was conducted. The postoperative patients of cardiac surgery and non-cardiac surgery who were transferred to ICU immediately, and who were at least 18 years old and whose ICU lengths of stay were at least 1 day, and who were admitted to ICU of the First Affiliated Hospital of Zhengzhou University from September 2014 to September 2016 were enrolled. According to the mean lactate concentration, the patients were divided into normal lactate group (0-2 mmol/L), relatively high lactate group (2-4 mmol/L), and absolute high lactate group (> 4 mmol/L), and the relationship between the mean lactate concentration and the prognosis of patients was analyzed. According to the degree of lactate variability, the patients were divided into four groups, and multivariate regression models were used to assess the risk of death in three different lactate variability groups. The value of the first lactate value and the highest lactate value during the first 24 hours in ICU were evaluated to predict the prognosis by the receiver operating characteristic (ROC) curve. Results 268 postoperative patients of cardiac surgery and 281 cases of non-cardiac surgery were selected, and the characteristic of the baseline data in the two groups was balanced. ① Mean lactate concentration and mortality in ICU: in the normal lactate group (0-2 mmol/L), there was no significant difference in mortality between the post-cardiac operative group and post-non-cardiac operative group [7.9% (14/177) vs. 6.5% (14/217), odds ratio (OR) = 1.245, 1 = 0.694]. In the relatively high lactate group (2-4 mmol/L), there was no significant difference between the two groups, either [33.3% (12/36) vs. 23.7% (9/38), OR = 1.611, 1 = 0.442]. In the absolute high lactate group (> 4 mmol/L), ICU mortality in post-non-cardiac operative group was obviously higher than that of post-cardiac operative group [69.2% (18/26) vs. 43.6% (24/55), OR = 0.344, 1 = 0.036]. ② The ranges of lactate variability per quartile (mmol·L-1·d-1) and ICU mortality risk: there was a linear relationship between lactate variability and ICU mortality in post-non-cardiac operative group, < 0.50 (reference), 0.50-0.85 (OR = 1.17, 1 = 0.87), 0.85-1.44 (OR =4.86, 1 = 0.04), > 1.44 (OR = 22.66, 1 < 0.01) , and there was a significant difference between the two groups in the high degree of variability (0.85-1.44 and > 1.44). The risk of death after cardiac surgery tended to increase, < 0.55 (reference), 0.55-1.25 (OR = 0.61, 1 = 0.61), 1.25-2.43 (OR = 3.46, 1 = 0.10), > 2.43 (OR = 12.14, 1 < 0.01), and the risk of death only showed difference in the highest degree of variation (> 2.43). ③ ROC curve showed that the area under ROC curves (AUC) of the highest lactate in 24 hours were larger than that of the first lactate in both groups, with higher sensitivity and specificity. In the post-cardiac operative group and post-non-cardiac operative group, the AUC of the highest lactate in the first 24 hours were 0.877 and 0.875, the cut-off values were 5.35 mmol/L and 5.65 mmol/L, the sensitivity were 81.4% and 67.9%, and the specificity were 93.8% and 96.1%, respectively. Conclusions Patients with post-non-cardiac operation should be more active in controlling hyperlactatemia and lactate variability. The highest lactate in the first 24 hours maybe one of the indicator for the assessment of the prognosis of the postoperative patients.