Changes Of TLR4 And Treg In Severely Burnt Rats And Effects Of Forsythia Suspense On Them

Gram-negative bacterial sepsis with resulting multiple organ dysfunction syndrome (MODS) and death continues to be a major problem in critical surgical patients. Lipopolysaccharide (LPS), an integral component of the gram-negative bacterial cell membrane, is responsible for many. At the early stage of severe burns, burn injury could result in the translocation of gut-derived endotoxin and bacteria, which triggers both excessive inflammation and suppressed adaptive immunity. TLR4 is a critical element of the mamaliar endotoxin receptor, it is a trans-membrane protein that initiates a signalling cascade tha triggers an innate immune response to endotoxin, it plays an essential role in innate host defense as well as in the control of adaptive immune responses, it evolved to detect the presence of infection through recognition of conserved pathogen-associated molecular patterns (PAMPs). CD4+CD25+ regulatory T cell (Treg ) is the cell which has unique immunologic characteristics compared with other immune cells, it plays a critical role in suppressing the development of autoimmune disease, in controlling potentially harmful infammatory responses, and in maintaining immune. Recently, the forkhead/winged helix transcription factor(Foxp3) was found to be unique expressed by Treg. Forsythia suspensa is a kind of commonly used Chinese herb which belongs to the category of treating hot evil, however, there is a few pharmacological researches about its anti-endotoxin and immunity modulation function and mechanism overall. The aim of this study was to explore the relationship among TLR4 on splenocytes, ET and Treg in peripheral blood of burnt rats during the early phase, roles of TLR4 in multiple organ dysfunction after severe burn and the effects of Forsythia suspensa on them.PartⅠ:Study on the expression of TLR4 on splenocytes and its relation with CD4+CD25+ regulatory T Cells in peripheral blood in severely burnt ratsObjective: To investigate the expression of Toll like receptor 4(TLR4) on splenocytes and TNF-αmRNA, the dynamic changes of CD4+CD25+ regulatory T cell(Treg) and endotoxin(ET) in peripheral blood of burnt rats during the early phase, and to explore the effects of endotoxemia on the immune system after burn.Methods: 64 SD male rats were randomly separated into control group and burn model groups. Rats of burn model group were burnt with vapor under 3mPa pressure and 108oC temperature for 8 seconds to achieve deep partial-thickness burn, and a 30% total body surface area(TBSA)burn model was made. Rats were sacrificed before and 2, 5, 8, 12, 24, 48 and 72 h after burn, and the TLR4 and TNF-αmRNA expression in splenocytes were measured at various intervals by RT-PCR. The expression of TLR4 protein was measured by Western blot, and the percentage of Treg cells in CD4+ T cells was detected by flow cytometry(FCM), and the LPS concentration of plasma was detected by limulus lysate test.Results: The expression of TLR4mRNA, TNF-αmRNA, TLR4 protein and the levels of Treg, ET were significantly increased at some times points after burn. The expression of TLR4mRNA and protein reached the peak at 8h, whereas the TNF-αmRNA at 12h, and Treg and ET at 8h. The peak values of them were of significant difference when compared with the control group (P<0.01). The expression of TLR4 mRNA showed a positive correlation with that of Treg, ET and TNF-αmRNA(P<0. 01). Conclusion: The up-regulation of LPS-TLR4 signal transduction maybe one of mechanisms of SIRS producing during the early phase after burn. The notable increase of Treg in peripheral blood resulting in the suppressing of immune system maybe one of mechanisms of bacteria translocation after severe burn. At the early stage of severe burns, the expression of TLR4 mRNA showed a positive correlation with the level of Treg, the up-regulation of LPS-TLR4 signal transduced by intestine-derived infection could lead to the increase of Treg.PartⅡ:The relationship and implication between the multiple organ dysfunction and TLR4mRNA expression in severely burnt ratsObjective:Through the detection of the specific expression of TLR4 and TNF-αin tissues of liver, heart, lung and small intestine, and the histopathology scores in them, to explore the role of TLR4 and its mechanism in multiple organ dysfunction after severe burn.Methods: Sixty-four SD rats were randomly divided into 8 groups as PartⅠ. Animals were sacrificed at various intervals, and TLR4mRNA and TNF-αmRNA expression in liver, lungs, hearts and small intestine were detected by reverse-transcription PCR. Protein of TLR4 activity also was evaluated by immunohistochemistry staining . The degrees of liver, lungs, hearts and small intestine damage were graded by the histopathology score.Results:1.The histopathology changes: The morphologic changes of internal organs after burn showed more severe than those in the control group,the liver injured first and badly.2.TLR4mRNA and protein expression : It was shown that the expression of TLR4 mRNA and protein in liver, lung, heart and small intestine were significantly increased after burn, the expressions of TLR4mRNA in liver and lung reached the peak at 8 h , whereas in heart and small intestine at 12 h, which were of significant difference when compared with the control group (P<0.01).3.TNF-αmRNA expression: It was shown that the expression of TNF-αmRNA in liver, lung, heart and small intestine were significantly increased in PBH groups, compared with the control group (p<0.01),they reached the peak at 12 h .4. Correlation analysis: The expression of TLR4 mRNA in liver, lung, heart and small intestine showed a positive correlation with that of TNF-αmRNA, P<0.01, also showed a positive correlation with the histopathology score of them, P<0. 01. Conclusion: Severe burn could lead to remarkable up-regulation of TLR4mRNA expression in vital organs, which might play an important role in the development of multiple organ dysfunction after severe burn.PartⅢ: The effects and mechanisms of Forsythia suspense on the expression of TLR4 on splenocytes and levels of ET,Treg in peripheral blood in severely burnt ratsObjective: To observe the effects of aqueous of Forsythia suspense(AFS) on the expression of TLR4,Foxp3 on splenocytes and levels of ET,Treg in peripheral blood of rats 8h after burn, and to observe the effects of AFS on the expression of TLR4,TNF-αmRNA in macrophage RAW264.7 induced by LPS, to explore the anti-endotoxin and immunity modulation function of AFS and its possible mechanisms .Methods: Cultured RAW264.7 cells have been randomly divided into six groups: control group: without any treatment; LPS group: adding LPS at final concentration of 10ng/ml for 16h; AFS1+LPS,AFS2+LPS,AFS3+LPS group: 30mins of AFS (50mg/ml,25mg/ml,12.5mg/ml in final concentration) preconditioning before adding LPS; Ploy B+LPS group: 30mins of Ploy B (10ug/ml in final concentration) preconditioning before adding LPS. The two intracorporal experiments were divided into six groups and five groups, respectively; the rats of AFS1 guoup,AFS2 group and AFS3 group of them were given AFS 5g/kg,2.5g/kg,1.25g/kg once a day by Po. pathway for seven days before burns, respectively. The cell viability of different groups were measured by MTT analysis; The expression of TLR4,TNF-αand Foxp3mRNA on RAW264.7 and splenocytes were measured by RT-PCR, respectively; The expression of TLR4 protein on RAW264.7 and splenocytes were measured by western blot; and the percentage of Treg cells in CD4+ T cells was detected by flow cytometry(FCM); and the LPS concentration of plasma was detected by limulus lysate test; protein of Foxp3 activity was evaluated by immunohistochemistry staining .Results:1. Effects of AFS on TLR4 signal-transducing pathway in macrophage induced by LPS1.1 MTT analysis: Compared with control group, the cell viability of LPS group significantly decreased (P<0.01); AFS1+LPS,AFS2+LPS,AFS3+LPS group significantly enhanced the cell viability(P<0.01), which was dose-dependent; there was no significant difference between AFS1+LPS group and Ploy B+LPS group.1.2 TLR4 mRNA and protein expression: It was found that TLR4 mRNA and protein had a basic expression in RAW264.7 cells under normal condition; and it was increased significantly after LPS acted on RAW264.7 cells than that of control group at 16h; adding AFS before the presence of LPS, the expressions of TLR4 in AFS1+LPS guoup,AFS2+LPS group and AFS3+LPS group decreased remarkably than that of LPS group, which was dose-dependent.1.3 TNF-αmRNA expression in RAW264.7 significantly increased in LPS group compared with the control group (p<0.01); AFS1,AFS2,AFS3 significantly decreased TNF-αmRNA expression (P<0.01), which was dose-dependent;there was no significant difference between AFS1+LPS and Ploy B+LPS group.2. Effects of AFS on the plasma level of ET and the expression of TLR4 on splenocytes of burnt ratsCompared with control group, mRNA expressions of TLR4 and TNF-αon the splenocytes were upregulated markedly(P<0. 01) and ET were significantly increased in the 8PBH (post burn hour-2) group. The protein expression of TLR4 on the splenocytes was consistent with the mRNA expression. AFS1,AFS2,AFS3and Ploy B significantly attenuated these increases, there was no significant difference between AFS1group and Ploy B group.3. Effects of AFS on the plasma level of Treg and the expression of Foxp3 on splenocytes of burnt ratsCompared with control group, the expression of Foxp3mRNA and protein on the splenocytes were upregulated markedly(P<0.01), and the percentage of Treg were significantly increased(P<0.01) in the 8PBH group. AFS1,AFS2 and AFS3 significantly attenuated these increases(P<0.01), which was dose-dependent. Conclusions: AFS preconditioning can inhabit the up-regulation of TLR4 in macrophage induced by LPS, and can decrease levels of ET,Treg in peripheral blood of severely burnt rats, and can down-regulate the expression of TLR4 in severely burnt rats; the anti-endotoxin and immunity modulation function and mechanism of AFS is firstly studied from the level of entire animal, cell and molecure,...