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BACKGROUND:Diabetic nephropathy is an important cause of end-stage renal disease,and intestinal barrier damage plays an important role in the occurrence and development of diabetic nephropathy. OBJECTIVE:To observe the protective effect of human umbilical cord mesenchymal stem cells on the intestinal barrier in rats with diabetic nephropathy. METHODS:Thirty 8-week-old male SD rats were randomly assigned to healthy control group,model group and human umbilical cord mesenchymal stem cell group,with 10 rats in each group.Rats in the human umbilical cord mesenchymal stem cell group were injected with 1×106 human umbilical cord mesenchymal stem cells through the tail vein once a week for 4 weeks after the model establishment of diabetic nephropathy.Rats in the healthy control group and the model group were injected with an equal volume of PBS at the same time.1 week after the last injection,the histomorphological changes in the kidney and colon were observed under a light microscope.The expressions of ZO-1 and Occludin in the colon tissue of rats were detected by immunohistochemistry.Serum D-lactic acid and lipopolysaccharide levels were detected by ELISA.In addition,the distribution of human umbilical cord mesenchymal stem cells labeled with DiR dye in rats was observed by in vivo imaging system.The expression of human mesenchymal stem cell surface marker antigens CD44 and CD90 in colon tissue was detected by immunohistochemistry. RESULTS AND CONCLUSION:(1)Compared with the model group,human umbilical cord mesenchymal stem cell transplantation significantly inhibited the increase of urea nitrogen,serum creatinine,24-hour urine protein level and urinary albumin/creatinine ratio in diabetic nephropathy rats(all P<0.05).(2)The expression of human mesenchymal stem cell surface markers CD44 and CD90 was found in the colon of diabetic nephropathy rats.(3)Compared with the healthy control group,the expression levels of tight junction proteins Occludin and ZO-1 in the colon tissue of the model group were significantly reduced,while the expressions of Occludin and ZO-1 were significantly increased after treatment with human umbilical cord mesenchymal stem cells.(4)Compared with the model group,human umbilical cord mesenchymal stem cell transplantation significantly reduced serum D-lactic acid and lipopolysaccharide levels in diabetic nephropathy rats.(5)The results suggest that human umbilical cord mesenchymal stem cells may protect the intestinal barrier function by enhancing the expression of intestinal tight junction proteins in diabetic nephropathy rats.
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OBJECTIVE@#To investigate the effect of Porphyromonas gingivalis (Pg) infection on IFNGR1 palmitoylation and biological behaviors of esophageal squamous cell carcinoma (ESCC) cells and the clinical implications.@*METHODS@#The expression levels of IFNGR1 protein in ESCC cell lines KYSE30 and KYSE70 were detected using Western blotting at 24 and 48 h after Pg infection, and 2-BP was used to detect IFNGR1 palmitoylation in the cells. KYSE70 cells with wild-type IFNGR1 (IFNGR1-WT cells) and with IFNGR1-C122A palmitoylation site mutation induced by site-specific mutagenesis (IFNGR1-C122A cells) were both infected with Pg, and the changes in palmitoylation of IFNGR1-C122A were analyzed using immunofluorescence and Click-iT assays. The changes in proliferation, migration and invasion ability of the infected cells were evaluated using plate cloning assay, scratch assay and Transwell assay, and IFNGR1 co-localization with lysosomal marker LAMP2 was dected using immunofluorescence assay. Immunohistochemistry was used to detect Pg infection and IFNGR1 protein expression in 50 ESCC tissues, and their correlation with the clinicopathological characteristics and survival outcomes of the patients was analyzed.@*RESULTS@#Pg infection down-regulated the protein expression of IFNGR1 in ESCC and promoted IFNGR1 palmitoylation at site 122. In IFNGR1-WT cells, Pg infection significantly enhanced cell proliferation, migration and invasion (P < 0.05). Similarly, Pg also significantly promoted proliferation, migration and invasion of IFNGR1-C122A cells, but to a lesser extent as compared with the wild-type cells (P < 0.05). Immunofluorescence assay showed that Pg and ZDHHC3 promoted IFNGR1 degradation within the lysosome. Immunohistochemical studies of the ESCC tissue samples showed a negative correlation between IFNGR1 and Pg expression, and a reduced IFNGR1 expression was correlated with a poorer survival outcome of the patient.@*CONCLUSION@#Pg infection enhances IFNGR1 palmitoylation to promote progression of ESCC, and elimination of Pg and inhibiting IFNGR1 palmitoylation may effectively control ESCC progression.
Subject(s)
Humans , Esophageal Neoplasms , Porphyromonas gingivalis , Lipoylation , Esophageal Squamous Cell Carcinoma , LysosomesABSTRACT
Objective @#To explore the relationship between Porphyromonas gingivalis(Pg) and mitophagy in esopha- geal cancer cells，and to explore new therapeutic targets for esophageal cancer．@*Methods @#① Western blot was used to detect the phosphorylation of unc-51-like authophagy activating kinase1 (ULK1) in mitochondria of the Pg infected cells and immunohistochemical method was used to detect the correlation between the expression of Pg and the phosphorylation status of ULK1 in esophageal cancer tissues. ② Western blot，ICC and ELISA were used to de- tect the transfer of nucleotide blinding domain and leucine rich repeat containing family member X1 (NLRX1) from cytoplasm to mitochondria，mitophagy，and the secretion levels of interleukin ( IL) -6 and reactive oxygen species (ROS) under Pg infection. ③ Pg colonization in esophageal tissues of mice in each group was detected by qPCR and Pg colonization in esophageal squamous epithelial cells of mice by RNAscope. @*Results @#Compared with the un- treated group，the phosphorylation level of mitochondrial ULK1 (P＜0．01) ，NLRX1 expression (P＜0. 001) and mitophagy (P＜0. 001) of esophageal cancer cells increased after Pg infection．Compared with the control group， the combined intervention group could inhibit Pg colonization in esophageal tissue and esophageal squamous epithe- lial cells of mice (P＜0. 001) ．@*Conclusion @#Pg promotes the translocation of NLRX1 from cytoplasm to mitochon- dria by up-regulating the phosphorylation level of ULK1 in the mitochondria of esophageal cancer cells，and then induces mitophagy，leading to the reduction secretion of IL-6 and ROS，and ultimately maintaining Pg colonization.
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OBJECTIVE：To shar e the experienc e of pharmaceutical care in Wuhan Jinyintan Hospital （herein after refers to “our hospital ”）under the condition of novel coronavirus pneumonia （COVID-19）epidemic，and to provide reference for other hospitals to deal with public health emergencies. METHODS ：The situation of pharmaceutical care in our hospital under the condition of COVID- 19 epidemic was summarized and shared ，including the epidemic prevention and control management （regional division ，disinfection management ，pharmacy personnel training ），supply of drugs and disinfection products ，the monitoring and education of rational drug use by information technology. RESULTS ：The pharmacy department of our hospital divided the activity scope into clean area ，potential pollution risk area ，semi pollution area ，and implement different disinfection management. All pharmacists received training ，involving personal health protection ，prevention and control knowledge of COVID-19，health status monitoring ，etc. For supply and guarantee of drugs and disinfectants ，the epidemic drug list of our hospital was formulated ，drugs and disinfectants were purchased accurately and stored in a standardized way. 24 h response telephone was set up in the clinical pharmacy room to receive consultation from clinicians on drug use at any time. The drugs mentioned in the COVID- 19 diagnosis scheme were compared in terms of the mechanism of action and the medication of special populations to form a tablet ，so as to help clinical rational choice treatment drug. CONCLUSIONS ：The pharmaceutical care in the designated hospital of COVID- 19 is a professional and complicated work ，involving a wide range of aspects. Pharmacy department must respond actively and adjust the strategy in time so as to play an important role in improving the ability of medical treatment.
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Objective To investigate the efficacy of small interfering RNA against Pseudomonos aeruginosa expressing MexA-MexB-OprM multidrug efflux pump in vivo.Methods Two short hairpin (sh)RNA expression vectors targeting the MexB gene,and negative controls,were designed,synthesized,and electrotransformed into the P.aeruginosa strain PAO1.The in vivo therapeutic efficacy of the MexB small interfering (si)RNAs was determined by infecting a murine model of chronic P.aeruginosa lung infection (1 × 107 CFU/ml).The mice were killed on day 3,5 and 7 after infection with the Pseudomonas aeruginosa strains.Results In the murine infection model,treatment with MexB-siRNAs led to significantly reduced bacteria burden of the bellows by day 5 and 7 post-infection,and reduced the P.aeruginosa-induced pathological changes.In addition,MexB-siRNA2 treatment enhanced neutrophil recruitment and production of inflammatory cytokines (IL-1β,IL-12) in the early infection stage (day 3) (P＜0.05),both of which decreased by day 7.Conclusion MexB-siRNA could inhibit both mRNA expression and the activity of P.aeruginosa in vitro.siRNA was effective in reducing the bacterial load in a murine model of chronic lung infection.Targeting of MexB with siRNA appears to be a novel strategy for treating P.aeruginosa infections.
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Objective To investigate the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in the lung tissue in a rat model of ventilator-induced lung injury. Methods Thirty healthy male SD rats weighing 200-250g were randomly divided into 3 groups( n = 10 each) : group A spontaneous breathing; group B small tidal volume (VT = 7 ml?kg-1, RR = 40 bpm, FiO2=0.21) and group C large tidal volume (VT = 40 ml?kg-1, RR = 20 bpm, FiO2=0.21). The animals were mechanically ventilated for 4 h in group B and C. The lung injury was assessed by measurement of PaO2 /FiO2 . At the end of the experiment the animals were killed and the lungs were removed. The right lung was used for determination of the expression of mRNAs of MMP-2, MMP-9, TIMP-1 and TTMP-2 using RT-PCR and histologic examination. The left lung was weighed and then lavaged. The broncho-alveolar lavage fluid (BALF) was collected for total WBC and neutrophil counts and determination of total protein content and gelatinase activity. After lavage the left lung was heated at 60℃. The W/D lung weight ratio was calculated. Results PaO2/FiO2 was significantly lower after 4h mechanical ventilation in group C than in group A and B. The total WBC count and total protein content in BALF were significantly higher in group C than in group A and B. The W/D ratio of the left lung was significantly higher in group C than in group A and B. Microscopic examination showed that there were marked WBC infiltration and destructive changes of alveolar walls in group C. The levels of gelatinase (MMP-2 and MMP-9) activities in BALF were significantly higher in group C than in group A and B. The expression of the mRNA of MMP-2 and MMP-9 was significantly higher in group C than in group A and B, but there was no significant difference in the expression of the mRNA of TIMP-1 and TIMP-2 between group C and group A and B. Conclusion Large tidal volume ventilation can induce acute lung injury. MMP-2 and MMP-9 play an important role in the development of ventilator induced lung injury and the imbalance between MMPs and TIMPs contributes to the mechanism of ventilator-induced lung injury.