ABSTRACT
Objective:To evaluate immune response of murine peritoneal macrophage challenging by methicillin-resistant S.aureus(MRSA)after pretreatment with Pam3Csk4(TLR2 agonist).Methods: Murine peritoneal macrophage was pretreated with Pam3Csk4(1 μg/ml).Following pretreatment 12 h later,heat-killed MRSA( HK-MRSA) was added and incubated for another 2 or 6 hours.The protein and mRNA level of TNF-α, IL-6 and IL-1 were determined by ELISA and Q-PCR, respectively.To estimate phagocytosis of macrophage,HK-MRSA/MSSA labeled with FITC( FITC-HK-MRSA/MSSA) were added to well and incubated for 30 min.After washing 5 times with PBS,intracellular FITC-HK-MRSA was detected by flow cytometry.To estimate antimicrobal activity of macrophage,live MRSA and MSSA were added to well and incubated at indication time,the CFU of s.aureus was estimated via a 10-fold serial dilution on agar media.cDNA was further quantitative assessed using primers for mouse FCR-Ⅰ,FCR-Ⅲ,CR-1,CR-3,iNOS and LL37 by Q-PCR .Results: Compared with saline-pretreated cell, the protein and mRNA level of TNF-α, IL-6 and IL-1 were markely reduced, respectively.However, both the phagocytosis and antimicrobal activity to S.aureus were significantly increased in macrophages pretreated with Pam3Csk4.Further study found that the macrophages had higher FCR-Ⅰ,FCR-Ⅲ,CR-1,CR-3,iNOS and LL37 expression at 6 h and 12 h post-stimulation Pam3Csk4.Conclusion: The results suggest that Pam3Csk4 could activate murine antimicrobal activity of peritoneal macrophage challenging by methicillin-resistant Saureus via increasing opsonophagocytosis in depended antibodies, complements manners.The results suggest Pam3Csk4 probably be a novel immunotherapy candidate against MRSA.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of hydroxycamptothecin (HCPT) on proliferation and apoptosis of rat hepatic stellate cells (HSC).</p><p><b>METHODS</b>Rat HSC line (HSC-T6) and rat hepatocyte line (BRL-3A) were treated with different concentrations of HCPT (0, 0.008, 0.016, 0.031, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32 mg/L respectively) for 24 h. Cell proliferation was assessed by MTT colorimetric assay, apoptosis was detected with PI staging followed by flow cytometry, and by DNA ladder assay. The morphological change of apoptosis was observed under transmission electron microscopy (TEM).</p><p><b>RESULTS</b>MTT assay indicated that HCPT significantly inhibited the proliferation of HSC-T6 and BRL-3A in a dose-dependent manner. 24 h after the treatment with different concentrations of HCPT (0.25, 0.5, 1 mg/L), the apoptosis rate (13.46%+/-2.42%, 26.25%+/-5.65%, 47.05%+/-8.76%, respectively) in HSC-T6 was significantly higher than that in control cells (4.89%+/-1.80%, F = 34.24, P less than 0.01). 24 h after 0.5 mg/L HCPT treatment, cell shrinkage, nucleoli disappearance, chromatin condensation were found under TEM, and DNA ladder was demonstrated by agarose gel electrophoresis.</p><p><b>CONCLUSION</b>HCPT could significantly inhibit proliferation and induce apoptosis of HSC-T6 in a dose-dependent manner.</p>
Subject(s)
Animals , Rats , Apoptosis , Camptothecin , Pharmacology , Cell Line , Cell Proliferation , Hepatic Stellate CellsABSTRACT
<p><b>OBJECTIVE</b>To explore the influence of lanthanum chloride on the TNFalpha expression of murine peritoneal macrophages stimulated by lipopolysaccharide (LPS).</p><p><b>METHODS</b>Murine peritoneal macrophages (Mphi) were isolated, cultured and then stimulated by LPS. The influence of lanthanum chloride on the TNFalpha secretion and TNFalphamRNA expression of murine Mphi stimulated by LPS was determined by ELISA method and SYBR green fluorescence quantitative RT-PCR. Forty BALB/C mice were randomly divided into two groups and were treated by lethal dose of LPS and lanthanum chloride processed LPS, respectively. The mortality within 7 days was observed.</p><p><b>RESULTS</b>The TNFalpha secretion and TNFalphamRNA expression level of the Mphi from mice treated by lanthanum chloride processed LPS were obviously lower than those by LPS only (P < 0.01). The mortality of the mice treated by lethal dose of LPS which has been processed by lanthanum chloride was significantly lower than that by lethal dose of LPS only.</p><p><b>CONCLUSION</b>Lanthanum chloride possessed the capacity of lowering down the toxicity of LPS and inhibiting the TNFalpha secretion and TNFalphamRNA expression in murine Mphi stimulated by LPS.</p>
Subject(s)
Animals , Female , Male , Mice , Cells, Cultured , Gene Expression Regulation , Lanthanum , Pharmacology , Lipopolysaccharides , Pharmacology , Toxicity , Macrophages, Peritoneal , Cell Biology , Metabolism , Mice, Inbred BALB C , RNA, Messenger , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Necrosis Factor-alpha , Genetics , Bodily SecretionsABSTRACT
Objective To induce the apoptosis of human alveolar epithelial cells(A\-\{549\}) by the extraction of the second stage larvae of Ascaris lumbricoides and investigate the extraction concentration and inducing time related to the apoptosis. \ Methods\ Following to the results of Microculture Tetrazolium Test (MTT), five concentrations of the extraction of the second stage larvae were chosen to induce the apoptosis of A\-\{549\} cells. Meanwhile, control groups without the inducement were set up. For each group, observation was made at five time points since the start of inducement, to assess the existence of apoptosis and percentage of cells showing characteristics of apoptosis. HE stain and diphenylamine reaction methods were used to assess the cell apoptosis. Agarose gel electrophoresis of DNA and flow cytometry were also employed to confirm the apoptosis for some groups. \{\ Results \ \}Observations indicated that the apoptosis ratio of A\-\{549\} cells induced by the extraction at different concentrations were significantly higher than that of the control cells (P