| Objective:By constructing deep-burn rat model with a boiled brass comb, we try to mimic the pathophysiological changes of burn wound progression. And then, utilizing this model, we wish to observe the potential therapeutic effect of Abnormal Savda Munziq (ASMq) on the early burn wound progression post burn injury, and analyze the possible dose effect and mechanism of regulation. Methods:First,200-220g Sprague-Dawley (SD) rat was selected as research animal. After anaesthesia had been induced (sodium pentobarbital,50 mg/kg intraperitoneally), a custom-made rectangular brass comb (with a transverse section of approximately 18mm×10mm) was boiled in 100℃ water for 5min and then applied to the shaved skin surface of the rat dorsum for 20 s after anaesthesia had been induced (sodium pentobarbital,50 mg/kg intraperitoneally). A row of four bands of full-thickness burns (20mm×10mm) was created with three interspaces of uninjured skin (20mm×5mm), with the interspaces representing the zone of stasis according to prior stud. The animals in all groups were sacrificed by intraperitoneal administration of a pentobarbital overdose at various time points. One band of the interspace skin with 2mm of burned tissue on each side (20mmx9mm) was harvested from each burn model and stored in 10%formalin at 4℃ for subsequent histologic and immunofluorescence analysis, while the remaining two bands of interspace skin (without burn tissue; 20mmx5mm) from each burned rat were stored at-80℃ for ELISA, RT-PCR, and Western blot assays.Second, found a key time-point of burn wound progression for interference, we applied different doses of ASMq to models bylavage, and tried to observe its effects on the zone of stasis. Finally, we explored the possible pathways which may be involved in the regulation of ASMq on burn wound progression by utilizing some signal inhibitor. Results:In the part Ⅰ, according to results of general observation and HE staining, we observed that the deep burn insult could lead to hyperemia or even necrosis in the tissue of the zone of stasis, as well as a tendency to merge between two direct burn wound.In addition, during 72h after burn insult, the level of oxidative stress increased gradually with varied time, while the activities of inner antioxidant enzymes (GPx, SOD) decreased significantly in view of increased consumption of them (P<0.05). Thelevelof oxidative stress in wound tissue peaked at 48h after burn injury. Meanwhile, Xanthine (XO) and NADPH non-phagocytic cell oxidase 4 (Nox 4), as two key enzymes in the process of production of free radicals, upregulated significantly in different time points post burn (P<0.05). With respect to inflammatory response, the burn insult could bring about significant augmentation of inflammatory mediators (MPO, IL-1β, IL-6) in the zone of stasis in burned rats (P<0.05), and the burn 48h group showed the most obvious release of inflammatory mediatorsaccording to results from RT-PCR and immunohistochemistry staining. Moreover, the protein expression of activated NF-κB also presented a gradual elevation with time (P<0.05), and all time groups indicated significant variance comparing with sham group (P<0.05). Additionally, the amount of apoptotic cells in zone of stasis increased significantly post burn, while the expressions of the apoptosis-relative protein-cleaved cysteinyl aspartate specific proteinase (Cleaved Caspase 3/9, CC3/9) were also upregulated obviously in the wound tissue. In the part II, the result of general observation revealed that ASMq administration could effectively relieved the tissue injuries in the zone of stasis. The ASMq treatment dose-relatively lowered the level of MDA in the wound tissue, while the activities of inner antioxidant enzymes (GPx, SOD) were elevated markedly by ASMq application (P<0.05). Considering XO an d Nox4, ASMq could also decrease their expressions in wound tissues (P<0.05). In addition, ASMqtreatment could attenuate the release of inflammatory mediators (P<0.05), and decrease their distribution in the zone of stasis post burn. The activation of NF-kB was also increased with the application of ASMq (P<0.05). The high dose of AMSq showed the most typical effect on the induction of tissue inflammation, and LPS could reverse the protective effect of high dose ASMq.Consideringcell apoptosis, we observed the amount of apoptotic cells in the zone of stasis decreased significantly after ASMq administration, while the expression of CC3/9 also presented obvious downregulation (P<0.05). The effect of ASMq enforced with increasing doses, and the Erk inhibitor PD98059 could significantly reverse the improvement of cell apoptosis and decreased CC3/9 expressions after high-dose ASMqtreatment (P<0.05). In the part III, we investigated the possible signaling pathways which may be involved in the mechanism of the effect of ASMq. The results fromwestern blotting and immunofluorescence staining, we discovered that:1) both of medium and high-dose ASMq treatments could ameliorate the activation ofNF-κBmarkedly, and high dose showed the most significant effect (P<0.01); 2) The phosphorylation of Bad and the expressions of Bcl-xL and Cyto C could be also regulated by ASMq. All three doses of ASMq could obviously increase the phosphorylation of Bad (P<0.01), and a dose-relative effect could be observed (P<0.01). With respect to Bcl-xL, both medium and high dose ASMq could upregulate its expression in the wound tissue. Meanwhile, the burn-induced increase of Cyto C expression in the zone of stasis could be downregulated by the treatment of medium and high-dose ASMq. High dose presented an enforced effect comparing with other lower doses on Bcl-xL and Cyto C expressions. PD98059 inference could reverse the effect of high-dose ASMq application, which indicates Erk may play an important role in the mechanism of ASMq; 3) The previous researches indicates ASMq may be able to regulate Ras/Erk/p90RSK signaling cascade, which is the important upstream signaling of mitochondria-apoptotic pathway. According to the result of immunofluorescence staining, ASMq administration could augment the distribution of phosphorylated Erk (p-Erk) with increasing doses (P<0.01). Moreover, western blotting also indicated three doses of AMSq could further increase phosphorylation of Erk dose-dependently. PD98059 could significantly attenuate the activation of Erk by high-dose ASMq (P<0.01). In addition, medium and high-dose of ASMq could obviously upregulate the expressions of Ras, but PD98059 showed no effect on Ras expression. Even low-dose ASMq could increase the expression of p90RSK, and the effect of ASMq was enforced with increasing doses (P<0.05). PD98059 displayed a remarked effect to downregulate ASMq-induced augmentation of p90RSK expression (P <0.01). Conclusion:1) Deep Burn wounds usually present a dynamic progression in which the existing wound tissue deepens and expands via multiple pathophysiological mechanisms during the first few days after the burn injury. Its detailed mechanisms include oxidative stress injury, inflammatory cascade activation and apoptosis; 2) ASMq could dose-relatively attenuate the increased oxidative stress, inflammatory response and cell apoptosis in the zone of stasis, as well as ameliorate the histological injury in burn wounds; 3) The regulation of ASMq on burn wound progression was mediated mainly by XO/Nox4, Ras/Erk/p90RSK/mitochondria-apoptotic signalling pathway and inflamma-tion-realtive NF-κB signalling. |
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