BACKGROUND: Renal tubulointerstitial fibrosis is mainly featured as the accumulation of extracellular matrix (ECM) in renal interstitium. The tubular epithelial-myofibroblast transdifferentiation (TEMT) is important to the pathogenesy of renal tubulointerstitial fibrosis. OBJECTIVE: To examine the effects of sodium ferulate (SF) on TEMT, and ECM main components such as collagen Ⅰ, collagen Ⅲ and fibronectin, in rat renal tubular epithelial cellsinduced by transforming growth factor-beta 1 (TGF- β1)- DESIGN: Randomized and controlled experimental study based on cells. SETTING: Department of Kidney in West China Hospital of Sichuan University. MATERIALS: Rat renal tubular epithelial cells (NRK-52E) originated from American Type Culture Collection (ATCC), were offered by the laboratory of Department of Nephrology in Australian Monash Medical Center. Cell strain used in this study was cultured at the 36th passage. SF white crystal with water solubility and more than 98.0% purify, was from Chengdu Hengda Pharmaceutical Co., Ltd. Different concentrations of SF (125,250, 500μreel/L) were designed in this study. Rabbit anti-rat α-smooth muscle actin (α -SMA) was produced by Wuhan Boster Company. Enzyme-linked immunosorbent assay (ELISA) kit was the produced of Shanghai Senxiong Science and Technology Co.,Ltd. Human recombinant TGF- β1 was produced by R&D Company. DNA Engine OpticonTM real-time fluorescence quantitative polymerase chain reaction apparatus was the product of MJ Research Company. METHODS: Rat renal tubular epithelial cells (NRK-52E) cultured in vitro were divided into five groups. Control group was added with serum-contained DMEM; TGF-β1-induced group was added with TGF-β1 at final concentration of 5 ng/L; SF at different concentrations groups were added with 125, 250, 500 μ mol/L SF and TGF- β1 at final concentration of 5 ng/L,respectively. MAIN OUTCOME MEASURES: The contrast phase microscope, real-time fluorescence quantitative polymerase chain reaction and ELISA method were used to detect TEMT of NRK52E cells induced by TGF-β1 and levels of collagen Ⅰ, collagen Ⅲ and fibronectin in the supernatant. RESULTS: Morphology of NRK52E cells: Compared with control group, TGF-β1 could induce the transdifferentiation of NRK52E cells, showing fibroblast-like in morphology after 3 days, which were previously the typical road stone-like epithelial cells. In three different concentration SF groups, the morphologic transformation stimulated by TGF-β1 could be partly ameliorated in a dose-dependent manner. Expression of α-SMA mRNA: Compared with control group, 5 ng/L TGF- β1 enhanced expression of α-SMA at 6 hours, and reached a peak at 72 hours; SF depressed the expression in a dose-dependent manner at 72 hours (P < 0.05). Changes of ECM: After induced by 5 ng/L TGF- β1 for 72 hours, the levels of collagen Ⅰ, collagen Ⅲ and fibronectin in the supernatant increased significantly (P < 0.05), whereas SF decreased these levels in a dose-dependent manner (P < 0.05). CONCLUSION: TGF- β1 induces the TEMT, and promotes the secretion of collagen Ⅰ, collagen Ⅲ and fibronectin. SF can inhibit TGF- β1-induced TEMT In a dose-dependent manner.

Objective To investigate the biological effects of Pseudomonas aeruginosa quorum sensing molecule OdDHL on murine mast cells. Methods The molecule structure and purity of synthesized OdDHL were confirmed by mass spectrum or proton nuclear magnetic resonance (NMR) and high-performance liquid chromatography, respectively. Its biological activity was checked using a quorum sensing sensor bacterial strain. The viability, apoptosis and intracellular calcium changes of P815 cell line in response to different concentration of OdDHL were determined. Results The biological active OdDHL was synthesized successfully. OdDHL inhibited proliferation of P815 cells in a dose, and time dependent manner. It also induced apoptasis and intracellular calcium release in P815 cells. Conclusion Psendomonas aeruginosa quorum sensing molecule OdDHL induces apoptosis and intracellular calcium release in murine mast cell line P815.

BACKGROUND: Connective tissue growth factor (CTGF) is a kind of factor that can mediate downstream action of transforming growth factor-β 1 (TGF- β 1). The upregulation of connective tissue growth factor expression plays an important role in pathological changes of renal interstitial fibrosis.OBJECTIVE: To explore the effect of Sodium Ferulate on the expression of CTGF mRNA and protein in rats with unilateral ureteral obstruction (UUO) and pathological changes of renal interstitial fibrosis, and to compare with Losartan.DESIGN: Randomized and controlled animal trial.SETTING: Department of Nephrology, West China Hospital of Sichuan University, and College of Public Health, Sichuan University.MATERIALS: Twenty-four healthy adult male SD rats were selected from the Experimental Animal Center of Sichuan University. Sodium Ferulate was provided by Sichuan Hengda Pharmacy Co, Ltd (No. 050302); rabbit anti-rat CTGF by Santa Cruz; Western blotting by BioRAD, USA; DNA Engine OpticonTM real-time fluorescent quantitation PCR device by MJ Research, USA.METHODS: The experiment was performed at Research Laboratory of Clinical Medicine (grade BSL-1), College of Public Health, Sichuan University from May to December 2006. Twenty-four healthy rats were randomly divided into 4 groups (n=6): UUO model group, Sodium Ferulate group, Losartan group, and sham-operation group. According to the previous protocol, UUO models were established in UUO model group, Sodium Ferulate group, and Losartan group, and the other rats were subjected to sham operation. From the first day after UUO, Sodium Ferulate group was intragastrically (i.g.)administrated with 150 mg/kg/d Sodium Ferulate; Losartan group was administrated ig. with 20 mg/kg/d. Losartan; UUO and sham operation groups were administrated i.g. with matching normal saline. All rats were executed 14 days after UUO to harvest partial renal tissues. All experimental procedure was accorded with animal ethical standards.MAIN OUTCOME MEASURES: The mRNA and protein expressions of CTGF were quantified by real-time PCR and Western blot. The pathological changes of renal interstitial tissues were observed by hematoxylin/eosin (HE) and Masson staining.RESULTS: Twenty-four rats were included in final analysis. Fourteen days after UUO, CTGF mRNA and protein expressions in UUO model group were significantly increased compared with sham operation group, but the expressions in Sodium Ferulate group were significantly lower than model group (P < 0.05). Compared with Losartan treated group, there was no significant difference (P > 0.05). HE and Masson staining showed inflammatory cell infiltration and tubular and interstitial changes as well as collagen deposition in renal interstitial tissues on day 14 after UUO. Sodium Ferulate obviously improved the renal pathological changes in UUO rats (P < 0.05), and the effect was similar to Losartan (P > 0.05).CONCLUSION: Sodium Ferulate inhibits UUO-induced renal interstitial fibrosis. This action, similar to the effect of Losartan, might be due to downregulation of CTGF expression.

Objective To investigate the effects of NOD2 on inflammatory responses of macrophages to Staphylococcus aureus. Methods Real-time RT-PCR detected NOD2 gene expression of macrophages infected by S. aureus. Synthesis of siRNA against NOD2 and interfere with macrophages, observed the effects of NOD2 gene silencing to phagocytosis of 5. aureus, cytokine secretion, activation of nuclear transcription factors, cell apoptosis of the macrophages infected by S. aureus using F.I .IS A, flow cytometry etc. Results S. aureus infection of macrophages can cause increased expression of intracellular NOD2. NOD2 gene silencing of macrophage lead to the decreased ability of phagocytosis with S. aureus, the lower levels of cytokines secretion, deficiencies of NF-κB activation. S. aureus can cause macrophage apoptosis, with the apoptosis rate increased with time. Conclusion The intracellular pattern recognition receptor NOD2 play a key role in pathogen recognition, signal transduction, activation of nuclear transcription factors in the process of macrophages infected by S. aureus.

Staphylococcus aureus (S. aureus) is an important human pathogen which can cause a chronic condition with a high relapse rate despite the aggressive antimicrobial treatment. Recent studies showed that intracellular pattern recognition receptors (including NOD) in response to bacteria or bacterial products play a proinflammatory role by activating nuclear transcription factor-κB (NF-κB). But how NOD2 mediates the proinflammatory response to S. aureus in mast cells (MCs) is unclear. So, in this study, we attempted to examine the role of NOD2 in inflammatory responses of MCs to S. aureus. P815 cells (a mouse mast cell line) were cultured. Real-time PCR was used to detect the NOD2 mRNA expression in P815 cells during S. aureus infection. The siRNA against NOD2 gene was synthesized and transfected into S. aureus-infected P815 cells. By using the methods of ELISA and flow cytometry, the effects of NOD2 gene silencing on cell phagocytosis, cytokine secretion, NF-κB activation and cell apoptosis of the S. aureus-infected P815 cells were examined. It was found that S. aureus infection could increase the expression of NOD2 mRNA in P815 cells. NOD2 gene interference in P815 cells reduced the number of S. aureus engulfed by P815 cells, the level of cytokines and the activation of NF-κB. In addition, S. aureus could induce the apoptosis of P815 cells, but NOD2 gene silencing did not affect the cell apoptosis rate. Our data suggested that NOD2 plays a key role in pathogen recognition, signal transduction, and NF-κB activation in the inflammatory responses of MCs infected by S. aureus.

ObjectiveTo investigate the alteration of MexB protein expression by plasmid containing siRNA template strand was transformed into Pseudomonas aeruginosa.Methods siRNA molecules specifically against mexB were designed and ligated into pGPU6/GFP/Neo vector to construct the recombinant plasmid pGPU6/GFP/Neo-siRNA.MexB gene was cloned into expression vector pET22b+ to construct plasmid pET22b+/mexB,the recombinant expression plasmid was transformed into E.coli BL21 (DE3)plysS and protein MexB was induced to express,then purified protein MexB was used to prepare specific antibodies in rabbits.The plasmids with siRNA molecules specifically against mexB were transformed into wild type strain,clinical multiresistant strain and mexB overexpression strain of Pseudomonas aeruginosa by electroporation respectively,and the changes of the expression of MexB were detected in 8,12,24 h respectively by Western blot.ResultspGPU6/GFP/Neo-siRNA was constructed successfully.Protein MexB was expressed successfully and the rabbit polyclonal antibodies against MexB was prepared well.The gene silence of mexB by siRNA molecules was effective in the three strains of Pseudomonas aeruginosa,but it depended on the silencing time.ConclusionThe expression of MexB was reduced in 8 h and 12 h,but in 24 h,the expression was unchanged.

Objective To investigate the effect and mechanism of siHybrids technique on inhibiring the efflux pump gene mexB of Pseudomonas aeruginosa PAO1 in vitro. MethodsTargeting the efflux pump gene mexB of Pseudomonas aeruginosa PAO1 ,we designed and synthesized three siHybrids molecule and one negative scamble siHybrids molecule. Pseudomonas aeruginosa PAO1 were intervened by the siHybrids molecules in 50 nmol/L, respectively. And the experiments were made of control groups[blank and scamble (sc) -001]and intervened groups[siHybrids( si ) -001, siHybrids( si ) -002 and siHybrids(si) -003]of Pseudomonas aeruginosa PAO1. The targeting efflux pump gene mexB mRNA expressions of Pseudomonas aeruginosa PAO1, including all groups, were measured by real-time PCR in 12 h and 24 h after interference in vitro. Further, the minimal inhibitory concentration (MIC) of chlormycetinCP, erythrocin( EM ), levofloxacin ( L-OFLX), ceftazidime ( CAZ), meropenem (MER) to those groups were detected by using Mueller-Hinton broth dilution before and after interference. ResultsThe relative mexB mRNA amounts of Pseudomonas aeruginosa PAO1 intervened by different siHybrids were not much more different from each other after 12 h,but the expression of mexB mRNA of the intervened group ( si-001 ,si-002 ,si-003 ) was much lower than control groups after interference for 24 h. The relative mexB mRNA amounts, comparing 12 h with 24 h, we would find the blank control and negative control submit escalating trend. And the intervened control ,three different siHybrids were all with a downward tendency. However, in presence of 50 nmol/L siHybrids, the minimal inhibitory concentration(MIC) of CP, EM, L-OFLX, CAZ, MER to those controls were not much more different before and after interference. Conclusion On level of the mRNA expressions, siHybrids could inhibit the efflux pump gene mexB of Pseudomonas aeruginosa PAO1 in vitro, and within 24 h would be able to function effectively. Further, the effect was time-dependent.

Objective To investigate whether Pseudomonas aeruginosa pvdQ( PA2385 ) gene reveals altered antibiotic susceptibility under swarming conditions. Methods The plasmid pME6032 with pvdQ gene was constructed and identified, then transformed into Pseudomonas aeruginosa PAO1 by the electroporation, building pvdQ overexpression strain. Using the same method building pME6032-PAO1 strain.Bacteria were inoculated in LB overnight , measuring the colony diameter of the swarming zone . Results Strains of pvdQ overexpression was successfully constructed by real-time PCR. Comparison of two strains of the swarming motility of change in diameter: The result showed that PAO1 and pvdQ overexpression strains can both improve the antibiotics resistance. Swarmer cells of pvdQ overexpression strain exhibited a 2- to 4-fold increase in antibiotic resistance toward ceftazidime,ciprofloxacin, meropenem and polymyxin B compared to PAO1 on BM2-swarming agar plates. Conclusion pvdQ gene played an important role in elevating the antibiotics resistance, which through prarticipated in the swarmer cell differentiation.

Abstract： Objective　To retrospectively analyze the investigation and disposal of the COVID-19 outbreak caused by the transmission of the Omicron variant in infected imported cases, and provide basis for COVID-19 outbreak management. Methods　The description epidemiological method was used to describe the COVID-19 outbreak in Sanya City from March 31 to April 15, 2022. The propagation chain was mapped and the experience gained and shortcomings identified in emergency responses were analyzed. Results　The outbreak resulted in 95 reported locally transmitted COVID-19 cases with a incubation period M(P25, P75) of 4 (3, 5) d. In the 95 cases, the proportion of cases detected through close contact screening, centralized isolation, community screening, control area screening, active treatment (examination), and key population screening were 33.68%, 22.11%, 18.95%, 12.63%, 6.32%, 4.21% and 2.11%, respectively. The epidemic spread for 6 generations, causing 5 clusters of outbreaks and 12 cases of cluster disease. The epidemic affected 12 villages/neighborhood committees, 1 bar, 1 hospital, 1 small clinic, 1 farmer's market, 1 large shopping mall and 1 restaurant in 2 districts of Sanya City. The result of gene sequencing was Omicron variant BA.1.1. Through the immediate launch of emergency plans, nucleic acid and antigen testing, controlling close contact between infected persons and close contacts, suspending indoor business sites, central urban control, and temporary suspension, COVID-19 was controlled within 16 days. Conclusions　The transmission chain of this outbreak was clear and was caused by imported cases. Strengthening the management of the pass, doing a good job in information sharing and docking, timely screening for cases, screening, pushing, controlling high-risk groups, and implementing comprehensive control measures, can effectively prevent the spread of the epidemic, providing a reference for the control of epidemic situations in relevant scenarios.

Staphylococcus aureus (S. aureus) is an important human pathogen which can cause a chronic condition with a high relapse rate despite the aggressive antimicrobial treatment. Recent studies showed that intracellular pattern recognition receptors (including NOD) in response to bacteria or bacterial products play a proinflammatory role by activating nuclear transcription factor-κB (NF-κB). But how NOD2 mediates the proinflammatory response to S. aureus in mast cells (MCs) is unclear. So, in this study, we attempted to examine the role of NOD2 in inflammatory responses of MCs to S. aureus. P815 cells (a mouse mast cell line) were cultured. Real-time PCR was used to detect the NOD2 mRNA expression in P815 cells during S. aureus infection. The siRNA against NOD2 gene was synthesized and transfected into S. aureus-infected P815 cells. By using the methods of ELISA and flow cytometry, the effects of NOD2 gene silencing on cell phagocytosis, cytokine secretion, NF-κB activation and cell apoptosis of the S. aureus-infected P815 cells were examined. It was found that S. aureus infection could increase the expression of NOD2 mRNA in P815 cells. NOD2 gene interference in P815 cells reduced the number of S. aureus engulfed by P815 cells, the level of cytokines and the activation of NF-κB. In addition, S. aureus could induce the apoptosis of P815 cells, but NOD2 gene silencing did not affect the cell apoptosis rate. Our data suggested that NOD2 plays a key role in pathogen recognition, signal transduction, and NF-κB activation in the inflammatory responses of MCs infected by S. aureus.
Animals ; Cell Line ; Cytokines ; immunology ; Inflammation Mediators ; immunology ; Mast Cells ; immunology ; microbiology ; Mice ; NF-kappa B ; immunology ; Nod2 Signaling Adaptor Protein ; immunology ; Staphylococcus aureus ; physiology