5-Fluorouracil (5-FU) is a commonly used chemotherapy drug, but there is no effective prevention and treatment for the side effects of intestinal mucositis induced by 5-FU. Inulin is a type of fermentable dietary fiber, which is non-digestible, and can improve metabolic function by modulating intestinal microbiota. The current study is aimed at appraising the effect of inulin on 5-FU-induced intestinal mucositis in mice by non-targeted metabolomics. The results indicated that oral inulin significantly inhibited 5-FU-induced body weight loss and intestinal shortening in mice, inhibited NLRP3 inflammasome activation and IL-1 beta expression, and increased IL-10 and IgA expressions. Inulin could significantly reduce intestinal mucosal injury induced by 5-FU in mice and significantly affected the production of 27 different metabolites in mouse serum. These metabolites were closely related to glycerophospholipid metabolism and retinol metabolic pathways. The study provided a potential new method for the prevention and treatment of chemotherapeutic intestinal mucositis.

Background: The primary reason for the development of chemotherapeutic intestinal mucositis induced by 5-fluorouracil (5-FU) is the activation of macrophages in the mucosa. This study aimed to explore how berberine can alleviate inflammation in macrophages and the resulting intestinal mucosal inflammation in mice induced by 5-FU.Methods: Tohoku Hospital Pediatrics-1 (THP-1) cell inflammatory response and mouse intestinal mucositis were induced by 5-FU and treated with berberine. The levels of inflammation-related factors and autophagy related proteins in THP-1 cells were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting. The concentrations of double-stranded DNA (dsDNA) and interleukin-1 beta (IL-1 beta) in mice serum and the small intestine hematoxylin-eosin (HE) staining were used to evaluate intestinal mucosal damage. Immunoglobulin A (IgA), an indicator of mucosal immunity, was detected in mice serum by enzyme-linked immunosorbent assay (ELISA). We used quantitative polymerase chain reaction (qPCR) to detect the relative contents of four important strains (Bifidobacterium, Lactobacillus, Escherichia coli, and Enterococcus) in the colon contents of mice. Additionally, we employed liquid chromatograph-mass spectrometer/mass spectrometer (LC-MS/MS) technique to measure the concentrations of three main short-chain fatty acids (acetic acid, propionic acid, and butyric acid) in the mice's plasma. Furthermore, we employed ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometer (UPLC-Q Tof-MS) technique to analyze the non-targeted metabolomics of mouse serum.Results: Berberine has been found to inhibit the expressions of NOD-Like Receptor Thermal Protein Domain Associated Protein 3 (NLRP3), Caspase-1, and IL-1 beta in THP-1 cells (p < 0.05). Additionally, it effectively suppresses the expression of autophagy related proteins LC3 and Beclin-1 in THP-1 cells (p < 0.05). Furthermore, in a mouse model study, berberine significantly enhances the levels of beneficial bacteria Bifidobacterium and Lactobacillus, as well as the concentrations of three main short-chain fatty acids (SCFAs) in the plasma of mice (p < 0.05). Moreover, it reduced the concentration of pro-inflammatory factor dsDNA and increased the mucosal immunity index IgA concentration in blood (p < 0.05). The untargeted metabolomics results demonstrated that berberine could regulate the inflammatory response of mice by impacting the metabolism of taurine, glycerol phospholipid, arachidonic acid, and primary bile acid biosynthesis.Conclusions: Berberine has demonstrated its ability to effectively suppress the inflammatory reaction of THP-1 cells induced by 5-FU. Furthermore, it also influences the "intestinal flora-metabolite-inflammation" pathway by regulating the composition of the intestinal flora, increasing the production of SCFAs, reducing the expression of inflammatory factors, and preserving the structural integrity of the intestinal mucosa.

Photodynamic therapy (PDT) has become a promising method for tumor treatment due to its non-invasive and high spatiotemporal selectivity. However, PDT is still hindered by reactive oxygen species deficiency, because solid tumors feature a hypoxic microenvironment. PDT combined with hypoxia-activated chemotherapy drugs can effectively induce tumor death, overcoming the limitations of the sole PDT for the fight against hypoxia. Herein, we designed a nanosystem (PCe6AZOM) that enhances the release of hypoxia-activated drugs (AZOM) by PDT. Under hypoxic conditions, the azo bond of AZOM is cleaved by azo reductase, releasing highly cytotoxic AZOM and resulting in a significant increase in intratumor drug concentration. Meanwhile, the commercial photosensitizer Ce6 can aggravate the oxygen-poor state during the PDT process and further cause more AZOM release. Moreover, the cascade reactions in the nanosystem could activate singlet oxygen and enhance drug release through 660 nm light laser irradiation, contributing to more effective induction of tumor apoptosis and tumor growth retardation in vitro and in vivo.

5-Fluorouracil (5-FU) is a common anti-tumor drug, but there is no effective treatment for its side effect, intestinal mucositis. The inflammatory reaction of macrophages in intestinal mucosa induced by 5-FU is an important cause of intestinal mucositis. In this study, we investigated the anti-inflammatory effects of the three important short-chain fatty acids (SCFAs), including sodium acetate (NaAc), sodium propionate (NaPc), and sodium butyrate (NaB), on human mononuclear macrophage-derived THP-1 cells induced by 5-FU. The expressions of intracellular ROS, pro-inflammatory/anti-inflammatory cytokines, as well as the nuclear factor-κB/NLR family and pyrin domain-containing protein 3 (NF-κB/NLRP3) signaling pathway proteins were determined. Furthermore, the cell metabolites were analyzed by untargeted metabolomics techniques. Our results revealed that the three SCFAs inhibited pro-inflammatory factor expressions, including IL-1β and IL-6, when treated with 5-FU (p < 0.05). The ROS expression and NF-κB activity of 5-FU-treated THP-1 cells were inhibited by the three SCFAs pre-incubated (p < 0.05). Moreover, NLRP3 knockdown abolished 5-FU-induced IL-1β expression (p < 0.05). Further experiments showed that the three SCFAs affected 20 kinds of metabolites that belong to amino acid and phosphatidylcholine metabolism in THP-1 cells. These significantly altered metabolites were involved in amino acid metabolism and glycerolphospholipid and sphingolipid metabolism. It is the first time that three important SCFAs (NaAc, NaPc, and NaB) were identified as inhibiting 5-FU-induced macrophage inflammation through inhibiting ROS/NF-κB/NLRP3 signaling pathways and regulating glycerolphospholipid and sphingolipid metabolism.

Nephropathy injury is a prevalent complication observed in individuals with diabetes, serving as a prominent contributor to end-stage renal disease, and the advanced glycation products (AGEs) are important factors that induce kidney injury in patients with diabetes. Addressing this condition remains a challenging aspect in clinical practice. The aim of this study was to explore the effects of Lactiplantibacillus plantarum NKK20 strain (NKK20) which protects against diabetic kidney disease (DKD) based on animal and cell models. The results showed that the NKK20 can significantly reduce renal inflammatory response, serum oxidative stress response, and AGE concentration in diabetic mice. After treatment with NKK20, the kidney damage of diabetic mice was significantly improved, and more importantly, the concentration of butyrate, a specific anti-inflammatory metabolite of intestinal flora in the stool of diabetic mice, was significantly increased. In addition, nontargeted metabolomics analysis showed a significant difference between the metabolites in the mouse serum contents of the NKK20 administration group and those in the nephropathy injury group, in which a total of 24 different metabolites that were significantly affected by NKK20 were observed, and these metabolites were mainly involved in glycerophospholipid metabolism and arachidonic acid metabolism. Also, the administration of butyrate to human kidney- (HK-) 2 cells that were stimulated by AGEs resulted in a significant upregulation of ZO-1, Occludin, and E-cadherin gene expressions and downregulation of α-SMA gene expression. This means that butyrate can maintain the tight junction structure of HK-2 cells and inhibit fibrosis. Butyrate also significantly inhibited the activation of PI3K/Akt pathway. These results indicate that NKK20 can treat kidney injury in diabetic mice by reducing blood glucose and AGE concentration and increasing butyrate production in the intestine. By inhibiting PI3K pathway activation in HK-2 cells, butyrate maintains a tight junction structure of renal tubule epithelial cells and inhibits renal tissue fibrosis. These results suggest that NKK20 is helpful to prevent and treat the occurrence and aggravation of diabetic kidney injury.

BACKGROUND:Gastric cancer is a common malignant tumor of the digestive tract and the fourth leading cause of death from cancer-related diseases. In recent years, many studies have found that circular RNAs play an important role in cancer. Tumor-associated macrophages (TAMs) are also critical for tumor progression.OBJECTIVE:This study examined the role of circRNA_102191 in gastric cancer progression.METHODS:The relative mRNA levels were determined by qRT-PCR. Western blotting and ELISA were used to detect the protein levels. In vitro proliferation was assessed using CCK8 and clonogenic assays. The migration and invasion of cell lines were assessed by transwell-based assays. The interactions between molecules were detected using a luciferase reporter assay. M0 macrophages were induced with PMA. M1 macrophages were induced with LPS and IFN-γ, and M2 macrophages were induced with IL-4.RESULTS:The expression of circRNA_102191 was enhanced significantly in gastric cancer cell lines and clinical tumor tissues. CircRNA_102191 promotes gastric cancer cell progression by regulating miR-493-3p and its downstream target gene XPR1. CircRNA_102191 can enhance the EMT process of gastric cancer cells by promoting the M2 polarization of macrophages.CONCLUSION:CircRNA_102191 promotes the biological function of gastric cancer cells by regulating the miR-493-3p/XPR1 axis and M2 macrophage polarization.

This study aims to evaluate the effect of berberine-based carbon quantum dots (Ber-CDs) on improving 5-fluorouracil (5-FU)-induced intestinal mucositis in C57BL/6 mice, and explored the mechanisms behind this effect. Thirty-two C57BL/6 mice were divided into four groups: normal control (NC), 5-FU-induced intestinal mucositis model (5-FU), 5-FU + Ber-CDs intervention (Ber-CDs), and 5-FU + native berberine intervention (Con-CDs). The Ber-CDs improved body weight loss in 5-FU-induced mice with intestinal mucositis compared to the 5-FU group. The expressions of IL-1β and NLRP3 in spleen and serum in Ber-CDs and Con-Ber groups were significantly lower than those in the 5-FU group, and the decrease was more significant in the Ber-CDs group. The expressions of IgA and IL-10 in the Ber-CDs and Con-Ber groups were higher than those in the 5-FU group, but the up-regulation was more significant in the Ber-CDs group. Compared with the 5-FU group, the relative contents of Bifidobacterium, Lactobacillus and the three main SCFAs in the colon contents were significantly increased the Ber-CDs and Con-Ber groups. Compared with the Con-Ber group, the concentrations of the three main short-chain fatty acids in the Ber-CDs group were significantly increased. The expressions of Occludin and ZO-1 in intestinal mucosa in the Ber-CDs and Con-Ber groups were higher than those in the 5-FU group, and the expressions of Occludin and ZO-1 in the Ber-CDs group were more higher than that in the Con-Ber group. In addition, compared with the 5-FU group, the damage of intestinal mucosa tissue in the Ber-CDs and Con-Ber groups were recovered. In conclusion, berberine can attenuate intestinal barrier injury and oxidative stress in mice to mitigate 5-fluorouracil-induced intestinal mucositis, moreover, the above effects of Ber-CDs were more significant than those of native berberine. These results suggest that Ber-CDs may be a highly effective substitute for natural berberine.

One of the most prevalent malignant tumors of the digestive tract is gastric cancer (GC). Age, high salt intake, Helicobacter pylori (H. pylori) infection, and a diet deficient in fruits and vegetables are risk factors for the illness. A significant risk factor for gastric cancer is infection with H. pylori. Infecting gastric epithelial cells with virulence agents secreted by H. pylori can cause methylation of tumor genes or carcinogenic signaling pathways to be activated. Regulate downstream genes' aberrant expression, albeit the precise mechanism by which this happens is unclear. Oncogene, oncosuppressor, and other gene modifications, as well as a number of different gene change types, are all directly associated to the carcinogenesis of gastric cancer. In this review, we describe comprehensive H. pylori and its virulence factors, as well as the activation of the NF-κB, MAPK, JAK/STAT signaling pathways, and DNA methylation following infection with host cells via virulence factors, resulting in abnormal gene expression. As a result, host-related proteins are regulated, and gastric cancer progression is influenced. This review provides insight into the H. pylori infection, summarizes a series of relevant papers, discusses the complex signaling pathways underlying molecular mechanisms, and proposes new approach to immunotherapy of this important disease.

The activation of the monocyte-macrophage system and the damage to the renal and pancreatic tissue are common complications in patients with diabetes induced by hyper-glycemia. This study aimed to evaluate the effect and mechanism of butyrate (NaB), a metabolite of intestinal flora, on inhibiting the inflammatory response of human monocyte-macrophages (THP-1 cells) induced by high glucose and the damage of pancreatic and renal tissue in diabetic mice. The results showed that high concentration glucose significantly up-regulated the expressions of IL-1β, TNF-α, and NLRP3 in THP-1 cells and mouse spleen, and that NaB could inhibit the overexpression of those genes. The abundance of Beclin-1, LC3B and reactive oxygen species (ROS) in THP-1 cells is increased due to the high glucose concentration, and NaB can inhibit the genes responsible for upregulating the expression. In diabetic mice, vacuolar degeneration of renal tubules was observed. Then we observed that some of the epithelial cells of the renal tubules were exfoliated and some formed tubules. NaB could alleviate these pathological lesions, but NaB cannot alleviate pancreatic injury. Our results indicated that NaB could be used for the prevention and adjuvant treatment of diabetic kidney injury.

OBJECTIVES:Gastric cancer is a common cancer of the digestive system. Long non-coding RNA (lncRNA) plays an important role in the formation and development of gastric cancer. This study aims to investigate the effect of long non-coding lncRNA 114227 on biologic behaviors in gastric cancer cells.METHODS:The experiment was divided into 4 groups: a negative control (NC) group, a lncRNA 114227 small interference (si-lncRNA 114227) group, an empty vector (Vector) group, and an overexpression vector (OE-lncRNA 114227) group. The expressions of lncRNA 114227 in gastric mucosa and gastric cancer tissues, gastric mucosal epithelial cells and different gastric cancer strains were determined by real-time reverse transcription PCR (real-time RT-PCR).The proliferation were detected by CCK-8 assay in gastric cancer cells. The epithelial-mesenchymal transformation (EMT) was utilized by Transwell assay, scratch healing assay, and Western blotting in gastric cancer cells. The effect of lncRNA 114227 on proliferation of gastric cancer cells was detected by tumor bearing experiment in nude mice in vivo.RESULTS:The expression level of lncRNA 114227 in the gastric cancer tissues was significantly lower than that in the gastric mucosa tissues, and in 4 kinds of gastric cancer strains was all significantly lower than that in gastric mucosal epithelial cells (all P<0.01). In vitro, the proliferation and migration abilities of gastric cells were significantly reduced after overexpressing lncRNA 114227, and cell proliferation and migration were enhanced after silencing lncRNA 114227 (all P<0.05). The results of in vivo subcutaneous tumorigenesis in nude mice showed that the tumorigenic volume of the tumor-bearing mice in the OE-lncRNA 114227 group was significantly smaller than that of the Vector group, and the tumorigenic quality was lower than that of the Vector group (P<0.05), indicating that lncRNA 114227 inhibited tumorigenesis.CONCLUSIONS:The expression of lncRNA 114227 is downregulated in gastric cancer gastric cancer tissues and cell lines. LncRNA 114227 may inhibit the proliferation and migration of gastric cancer cells through EMT process.

Objective: The objective is to explore the mechanism of inhibitory effect of three main SCFAs (acetate, propionate and butyrate) on inflammatory response of A549 cells. Methods: Human lung adenocarcinoma cells (A549 cells) were cultured, and were divided into normal control group (NC group), A. baumannii infection group (A. baumannii group), NF-κB inhibitor group (JSH group), A. baumannii infection + sodium acetate group (NaAc group), A. baumannii infection + sodium propionate group (NaPc group) and A. baumannii infection + sodium butyrate group (NaB group). Real-time quan-titative PCR was used to detect the mRNA expression of NLRP3, Caspase-1, IL-1β, IL-6, and TGF-β in A549 cells. Western blotting assay was used to de-termine the expression of autophagy and “pyroptosis” related proteins of NRLP3, cleaved-Caspase-1 (P20), GSDMD (P30), LC-3 and Beclin-1. At the same time, the expression of NF-κB p65 protein in nucleus and cytoplasm of A549 cells was detected. The level of oxygen species induced by A. baumannii. Compared with A. baumannii group, the expression of NF-κB p65 in nucleus was significantly decreased and the expression of NF-κB p65 in cytoplasm was significantly increased after 24 h pre-incubation with NaB, NaPc and NaAc, respectively. Conclusion: A. baumannii can induce inflammatory injury of pulmonary epithelial cells, and the three major SCFAs can inhibit the activation of NLRP3 inflammasome and the release of pro-inflammatory factors through NF-κB/ROS/NLRP3 pathway, which provides a new way for clinical prevention of severe inflammatory injury caused by A. baumannii infection.

This study evaluated the anti-inflammation effect of the three main short-chain fatty acids (SCFAs) on Acinetobacter baumannii-induced THP-1 cells. The three main SCFAs could inhibit A. baumannii-stimulated THP-1 cell NF-κB pathway activity and the expressions of NLRP3 inflamma-some and GSDMD, and increase autophagy. The three main SCFAs, especially the sodium butyrate (NaB), had the effect of down-regulation of ROS and TLR-2 expression in THP-1 cells. NaB and sodium propionate (NaPc), but not sodium acetate (NaAc), dramatically suppressed IL-1β and IFN-γ expression. The results indicated that NaB and NaPc could significantly inhibit the inflammation of THP-1 cells induced by A. baumannii, and the inhibitory effect was in the order of NaB > NaPc > NaAC. NaB and NaPc may inhibit inflammation through TLR-2/NF-κB/ROS/NLRP3 signaling pathway.

目的 探讨不同浓度的丁酸钠抑制氧化型低密度脂蛋白(Ox-LDL)诱发人单核巨噬THP-1细胞炎症反应能力及其机制.方法 分别使用终浓度为50、100、200、400、800μmol/L的丁酸钠预处理THP-1细胞24 h,再以终浓度为50 mg/L的Ox-LDL刺激THP-1细胞24 h,采用实时荧光定量PCR法和ELISA法检测IL-1β、TNF-α、IL-10和TGF-β表达量,实时荧光定量PCR法和免疫印迹法检测NLRP3和Caspase-1的表达量,免疫印迹法检测自噬相关蛋白LC3-Ⅱ和Beclin-1的表达量,以及NF-κB p65在细胞核和胞浆中的分布情况.本研究同时设置正常对照组和无丁酸钠预处理的Ox-LDL组.结果 与Ox-LDL组比较,分别经终浓度为200、400、800μmol/L丁酸钠预孵育后,THP-1细胞IL-10和TGF-β表达量显著升高(P<0.01);50、100、200、400μmol/L丁酸钠预处理组细胞TNF-α表达量明显下降(P<0.01);50和100μmol/L丁酸钠预处理组细胞IL-1β表达量明显下降(P<0.01);50、100、200、400、800μmol/L丁酸钠预处理组细胞Caspase-1表达量明显下降(P<0.01);仅400和800μmol/L丁酸钠预处理组细胞NLRP3蛋白表达量明显下降(P<0.01),其他浓度丁酸钠抑制效果不明显.Ox-LDL刺激后THP-1细胞NF-κB p65蛋白明显由细胞浆移位至细胞核内(P<0.01),而经50、100、200、400、800μmol/L丁酸钠预处理后,NF-κB p65蛋白核转位现象明显受到抑制(P<0.01).与Ox-LDL组比较,200、400、800μmol/L丁酸钠可以显著上调THP-1细胞Beclin-1表达量(P<0.01),50、100、200、400、800μmol/L丁酸钠可以显著上调LC3-Ⅱ表达量(P<0.01).结论 丁酸钠可以抑制由Ox-LDL诱发的THP-1细胞炎症反应,其可能的机制是通过上调THP-1细胞自噬水平,抑制THP-1细胞NF-κB信号通路和NLRP3炎症复合体活化而产生抗炎效果.本结果为采用"肠道微生物组-免疫炎症轴"理论干预高血脂患者体内慢性非可控性炎症提供了实验依据.

5-Fluorouracil (5-FU) is a used chemotherapy drug for cancer, and its main side effect is intestinal mucositis which causes chemotherapy to fail. It was known that short-chain fatty acids (SCFAs) can inhibit immune cell release of various proinflammatory factors and inhibit excessive intestinal inflammation. However, the inhibitory effect of SCFAs on 5-FU-induced intestinal mucositis is still unclear. To simulate the effects of SCFAs on immune and intestinal epithelial cells, the cells (THP-1 cells and Caco-2 cells) were pretreated with sodium acetate (NaAc), sodium propionate (NaPc) and sodium butyrate (NaB), then inflammation was induced by 5-FU. The expressions of reactive oxygen species (ROS), Beclin-1, LC3-II, NF-κB p65, NLRP3 inflammasome, proinflammatory/anti-inflammatory cytokines and mucosal tight junction proteins were determined. In our results, the three SCFAs could inhibit ROS expressions, NLRP3, Caspase-1, IL-1β, IL-6, IL-18, Beclin-1 and LC3-II, when induced by 5-FU. In a 5-FU-induced chemoentermuctis mouse model, Lactobacillus rhamnoides can increase the concentrations of three SCFAs in faeces and increase the concentrations of IL-1β, IL-6 and IgA in serum, and decrease the expressions of NLRP3 and IL-17 in spleen cells. The expressions of ZO-1 and Occludin in intestinal mucosa were significantly increased. These results indicated that the three SCFAs can effectively suppress the inflammation of THP-1 cells and Caco-2 cells and maintain tight junction integrity in intestinal mucosal epithelial cells.

探讨肺炎支原体脂质相关膜蛋白(LAMPs)诱发THP-1细胞炎症反应机制.提取LAMPs并刺激THP-1细胞,ELISA法检测细胞培养液上清中IL-1β,IL-18和TNF-α浓度,qRT-PCR法检测NLRP3和Caspase-1基因表达量,免疫印迹技术检测NF-κB信号通路活化,流式细胞术检测活性氧(ROS)生成量.结果显示:LAMPs显著提高IL-1β,IL-18和TNF-α表达(P<0.05);NLRP3和Caspase-1基因表达量均显著升高(P<0.05);LAMPs刺激后胞核NF-κB p65蛋白表达显著升高(P<0.05);细胞ROS生成量显著升高(P<0.05).LAMPs可以通过ROS/NF-κB/NLRP3途径诱发宿主巨噬细胞炎症反应,且随浓度升高诱导炎症反应能力增强.

The overactivation of macrophages causes chronic inflammatory diseases. Short-chain fatty acids (SCFAs), potential drugs for clinical treatment, are modulators of macrophage inflammatory reaction. Therefore, the modulation of macrophage-mediated cell activity is expected to become a new therapeutic strategy for inflammatory diseases caused by Mycoplasma pneumoniae. In this study, 2 kinds of SCFAs (propionate and butyrate) were found to have anti-inflammatory effects in M. pneumoniae-stimulated THP-1 cells inflammatory. They inhibited the expressions of IL-4, IL-6, ROS, and NLRP3 inflammasome, while enhancing the expressions of IL-10 and IFN-γ. Our study revealed these 2 agents to repress transcriptional activities of NF-κB, which are important modulators of inflammation. Meanwhile, SCFAs can significantly enhance the autophagy induced by M. pneumoniae. Considering that SCFAs have few side effects, they might be the promising adjuvant therapy for the prevention and/or treatment of various inflammatory diseases.

目的 研究表达Panton-Valentine杀白细胞素(PVL)的金黄色葡萄球菌诱导人THP-1细胞自噬和凋亡机制.方法 在大肠杆菌中表达重组杀白细胞素(rPVL),并免疫家兔制备多克隆抗血清.采用反转录PCR和Western blot法鉴定表达PVL的耐甲氧西林金黄色葡萄球菌(MRSA).分别使用表达PVL的MRSA(MRSAPVL+)和不表达PVL的MRSA(MRSAPVL-)感染THP-1细胞,于感染后1、3、6h收集细胞,采用实时定量PCR检测自噬相关基因3(ATG3)、ATG4B、ATG5、ATG7、ATG12;Western blot法检测beclin-1、微管相关蛋白1轻链3(LC3)、磷脂酰肌醇3激酶(PI3K)、磷酸化的PI3K(p-PI3K)、蛋白激酶B(AKT)、磷酸化的AKT(p-AKT)、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化的mTOR(p-mTOR)、裂解型胱天蛋白酶3(c-caspase-3)、caspase-3的蛋白水平;二氯二氢荧光素二乙酸酯(DCFH-DA)荧光探针负载法检测细胞活性氧(ROS)水平;异硫氰酸荧光素标记的膜联素V/碘化丙啶(annexin V-FITC/PI)双标记结合流式细胞术检测细胞凋亡.结果 成功制备PVL多克隆抗体,鉴定出1株MRSAPVL-菌和1株MRSAPVL+菌.分别以两株菌与THP-1细胞共培养1h,MRSAPVL+组仅ATG7表达量显著高于MRSAPVL-组和正常对照组;共培养3h,MRSAPVL+组ATG7和ATG12表达量高于MRSAPVL-组和正常对照组,其他基因表达量变化不明显.感染1、3、6h,MRSAPVL+感染组细胞PI3K、AKT和mTOR磷酸化水平显著低于MRSAPVL-感染组和正常对照组;感染1h和3h,MRSAPVL-感染组细胞PI3K、AKT和mTOR磷酸化水平显著低于正常对照组;感染6h,MRSAPVL-菌感染组细胞mTOR磷酸化水平显著低于正常对照组.感染1、3、6h,MRSAPVL+组细胞ROS水平和凋亡率均高于MRSAPVL-组和正常对照组,且随着感染时间的延长细胞ROS水平和凋亡率持续增加;MRSAPVL-组细胞ROS水平和凋亡率也高于正常对照组.结论 MRSAPVL+对THP-1细胞PI3 K/AKT/mTOR信号通路抑制作用更强,可促进ATG12/ ATG7/ATG5自噬通路活化增加THP-1细胞自噬;同时,MRSA PVL诱导THP-1细胞ROS产生增加,促进细胞凋亡.

目的 研究我院分离鲍曼不动杆菌携带blaOXA-23基因的携带情况及其诱导细菌耐药机制.方法 收集江阴市青阳医院2019年1月至2019年12月间临床分离鲍曼不动杆菌,经VITEK-2全自动微生物鉴定及药敏分析系统鉴定耐亚胺培南菌株100株,敏感菌株100株.通过多重PCR法检测上述菌株中blaOXA-23、blaOXA-24、blaOXA-51和blaOXA-58基因携带情况.通过PCR法扩增亚胺培南耐药的鲍曼不动杆菌blaOXA-23的ORF序列,构建过表达载体blaOXA-23/pYMAb3并电转化鲍曼不动杆菌(ATCC 17978),以硫酸卡那霉素和美罗培南共同筛选过表达bla OXA-23基因的菌株.采用稀释法检测转化blaOXA-23/pYMAb3载体和pYMAb3空载体的菌株对亚胺培南的MIC.结果 我院耐亚胺培南的鲍曼不动杆菌均携带blaOXA-23,敏感株未检出携带该基因.RT-PCR法可检出转化blaOXA-23/pYMAb3载体菌株表达blaOXA-23基因,而转化pYMAb3空载体菌株未检出blaOXA-23基因表达.转化blaOXA-23/pYMAb3载体菌株对亚胺培南MIC为32 μg/mL,转化空载体菌株MIC仅为0.5 μg/mL.结论 我院耐亚胺培南的鲍曼不动杆菌均携带blaOXA-23基因,可以作为耐药菌株筛选的分子标记.由质粒携带的blaOXA-23基因是诱发我院鲍曼不动杆菌耐药的重要原因,在院感工作中需高度重视.

目的:研究3种主要短链脂肪酸(short-chain fatty acids,SCFAs)即乙酸钠、丙酸钠和丁酸钠,对5-氟尿嘧啶(5-Fluorouracil,5-FU)诱发人单核巨噬THP-1细胞炎症反应的影响及其可能机制.方法:将THP-1细胞分为5组:对照组,细胞未经任何处理;5-FU组,2.5 mmol/L 5-FU处理细胞24 h;5-FU+乙酸钠组、5-FU+丙酸钠组、5-FU+丁酸钠组,均经100μmol/L相应SCFAs预处理24 h,再加入2.5 mmol/L 5-FU处理24 h.采用qRT-PCR法检测细胞炎症相关因子mRNA表达量;ELISA法检测细胞上清液中IL-1β、IL-6和IL-10含量;蛋白质印迹法检测细胞Toll样受体9(TLR9)、NOD样受体家族3(NLRP3)、Caspase-1、LC3-Ⅱ、Beclin-1表达量,以及细胞核和胞质中核因子-κB(nuclear factor kappa B,NF-κB)p65蛋白表达量;流式细胞术检测细胞活性氧水平.结果:与对照组相比,5-FU组TLR9、NLRP3、Caspase-1、IL-1β、IL-6、LC3-Ⅱ、Beclin-1、胞核NF-κB p65蛋白表达量以及活性氧水平显著增加(P均<0.01).与5-FU组比较,5-FU+乙酸钠组TLR9表达量显著降低(P<0.01);5-FU+丙酸钠组IL-1β表达量以及活性氧水平显著降低(P<0.01);5-FU+丁酸钠组IL-1β表达量以及核内NF-κB p65蛋白表达量显著减少(P<0.01),而胞质内表达量显著增加(P<0.01);3种SCFAs预处理均可以显著降低NLRP3、Caspase-1、IL-6、LC3-Ⅱ及Beclin-1表达量(P<0.01),并增加抗炎因子IL-10表达量(P<0.01).结论:乙酸钠、丙酸钠和丁酸钠3种SCFAs可以显著抑制由5-FU诱发的THP-1细胞炎症反应,并抑制细胞自噬水平,减少细胞活性氧生成量,其可能是通过抑制TLR9/NF-κB/ROS途径产生抗炎作用.

Objective: To investigate the drug of Bacillus licheniformis Capsule (BLC) to improve the high levels of cytokines and clinical prognosis in COVID-19 patients, and to provide the clinical evidence for gut microflora therapy in COVID-19.