| Objective Severe burned patients are often suffered from respiratory dysfunctions, which is usually difficult to control and is the main cause of death. Previous studies have found that lung injury and respiratory muscle atrophy were two main reasons leading to respiratory dysfunctions. Apoptosis plays an important role in burns-induced lung injury, meanwhile, apoptosis has also been shown as a key mechanism of skeletal muscle atrophy of limbs after severe burns. Thus, based on those findings, the present study was to investigate the following two aspects:1. The changes and possible regulatory mechanisms of apoptosis in rat lung tissue after severe burns and its effects on lung tissues; 2. The characteristics of apoptosis in rats diaphragm and the potential regulatory role of death receptor pathway.Methods 1. According to a random digital table, a total number of 32 W is tar rats were divided into 4 groups:sham burn group (SB group), burn group (B group),12-week post burn recovery group (12WR group), and 12-week post burn recovery plus a second burn injury group (12WB group). In SB and B groups, lung tissues were harvested on post burn days 4. The 12WR group and 12WB group received first Burn injury and 12 weeks later received separately a second sham burn injury and burn injury. Lung tissues were harvested on post burn days 4 after the second burn injury. All tissues were examined for cells apoptosis by TUNEL. Pulmonary fibrosis were assessed by Masson trichrome staining and Sirius red staining. The protein expression levels of Bcl-2,Bcl-xl, Bax, Cleaved Caspase-3, YAP, P-YAP, MST, P-MST, LATS1 and LATS2 were assessed by Western Blot.2.80 male Wistar rats were randomly divided into 2 groups:sham burn injury group (n=40) and severe burn injury group (n=40).After an overnight fasting, animals were euthanized at 0,1,4,7,10 days after the injury, blood was drawn from abdominal aorta and diaphragm was completely excised and their wet weights was recorded. Using transmission electron microscopy (TEM) to observe the ultrastructure of diaphragmatic apoptosis; the location of Caspase-3 was detected by immunohistochemistry; using enzyme-linked immunosorbent assay (ELISA) to measure serum sFas, sFasL, and sTRAIL levels; the protein expression of Akt,P-Akt, Bax, Bcl-2, caspase-8 and caspase-3 were examined by Western blotResults 1. Both Masson trichrome staining and Sirius red staining showed obvious pulmonary fibrosis in 12WR group and 12WB group. The apoptosis rates of B,12WR and 12WB group were significantly higher than that in group A (all P<0.05). Compared to SB group, cleaved Caspase-3, P-YAP/YAP, Bax/Bcl-2, P-MST, MST and LATS1 levels were significantly higher in B,12WR and 12WB group (all P<0.05). The P-YAP, which was detected by immunohistochemistry, increased in B,12WR and 12WB group.2. Burn injury resulted in significant reduction of body weight, diaphragm mass, and CSA. TEM observation revealed apoptotic myonuclei in burn injury group, which were characterized by irregularly condensed chromatin aggregated close to the myonuclear membrane. Ratio of sFasL/sFas and serum sTRAIL in burn injury group were increased compared with that of time-matched sham group. Western Blotting showed that:P-Akt on day 1,4 were significantly decreased while Bax/Bcl-2 on day 1,4,7,cleaved caspase-8 expression on day 1,4 were significantly increased in burn injured group. Cleaved caspas-3 reached peak on day4 in burn injured group.Conclusion 1. Severe burn injury could induce apoptosis in rat lung tissue. The changes of Hippo pathway and its downstream YAP were consistent with apoptosis. Hippo signaling pathway may play an important role in the regulation of apoptosis in severe burned rats lung tissues.2. Severe burn injury could induce atrophy in diaphragm in rats, which was supported by decreased diaphragm mass and muscle CSA. Apoptosis is an important cause of atrophy, wherein the death receptor pathway is an potential regulatory mechanism. |
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