| Part1 The dynamic change of mitochondrial dynamics and apoptosis in LPS-induced ALI in miceBackground The incidence of liver injury in sepsis is high, its pathogenesis is complex and has an important relationship with the prognosis of sepsis. Numerous studies found that mitochondria dynamics closely related with mitochondrial function and apoptosis and play an important role in the development of liver injury.Objective To observe the dynamic changes of mitochondrial dynamics,mitochondrial function and apoptosis in LPS-induced liver injury in different periods.Methods 60 C57BL/6 mice were randomly divided into six groups:normal group, 1 hour group, 3 hours group, 6 hours group, 12 hours group and 24 hours group, each group had 10 mouse, intraperitonealled LPS30mg/kg to made the model, after LPS injection corresponding time,taken blood to test the liver function, gross morphology of the liver, HE stained of liver tissues were analyzed; used the ELISA to assay of the ATP yield, MRCCI and Cyt-c content liver tissue and used Real-TimePCR to test the Drp1/Mfn2/Bcl-2/Bax / Caspase-3 m RNA levels of the liver tissue.Results(1) Compared with the normal group, the serum ALT levels(81.85±12.07 vs 55.58±11.64, p <0.01), AST levels(295.28±25.83 vs157.90±52.97, p <0.01) in mice of 3h group increased significantly; the mitochondria ATP and MRCCI(36.09 ±12.88 vs 49.58±19.79 and24.02±13.96 vs 28.06±10.86, p>0.05) decreased, but the difference was not statistically significant, but Cyt-c(4.87±0.65 vs 0.55±0.23, p<0.01)were significantly higher; the pathological damage index of liver(0.22±0.10 vs 0, p <0.05) significantly increased. Compared with the normal group, the serum ALT level(96.28±9.71 vs 55.58±11.64, p<0.01),AST levels(484.80±175.33 vs 157.90±52.97, p <0.01) of model group(6h group) was significantly higher; the mitochondrial ATP and MRCCI(23.99±10.2 vs 49.58±19.79 and 11.74 ±8.63 vs 28.06±10.86, p<0.05)decreased significantly, Cyt-c(6.49±1.07 vs 0.55±0.23, p<0.01) was significantly increased; the pathological damage index of liver(0.79±0.27 vs 0, p<0.05) significantly increased. Compared with the 6h group, the serum ALT levels(62.30±13.94 vs 96.28±9.71, p<0.01), AST levels(190.04±106.53 vs 484.80±175.33, p<0.01) of 12 h group decreased significantly; the mitochondrial ATP and MRCCI(40.09±8.71 vs23.99±10.2 and 25.84±10.25 vs 11.74±8.63, p<0.05) increased significantly, Cyt-c(3.39±0.39 vs 6.49±1.07, p<0.01) was significantly reduction;the pathological damage index of liver(0.36±0.18 vs 0.79±0.27, p<0.05)decreased significantly. Compared with the 6h group, ALT level(54.75±7.36 vs 192.03±27.33, p<0.01), AST levels(190.04±106.53 vs 484.80±175.33, p<0.05) of 24 h group decreased significantly; the mitochondrial ATP and MRCCI(45.53±16.69 vs 23.99±10.2 and 29.41±7.70 vs 11.74±8.63, p<0.01 and p<0.05) increased significantly, Cyt-c(1.89±0.58 vs6.49±1.07, p<0.01) was significantly reduced; the pathological damage index of liver(0.10±0.04 vs 0.79 ±0.27, p<0.05) decreased significantly.(2) Compared with the normal group, the Drp1 m RNA of liver tissue in3 h group(0.56±0.10 vs 0.46±0.15, p>0.05) increased, but the difference was not statistically significant; Mfn2 m RNA of liver tissue( 0.71±0.27 vs 1.15±0.30, p<0.01) decreased significantly; Bcl-2 m RNA(0.42±0.19 vs 1.13±0.25, p<0.01) decreased significantly; and Caspase3 m RNA and Bax m RNA(0.77±0.52 vs 0.52±0.11 and 0.56±0.22 vs 0.49±0.06, p>0.05)increased, but the difference was not statistically significant. Compared with the normal group, the Drp1 m RNA of liver tissue in 6h group(0.85±0.13 vs 0.46±0.15, p<0.01) significantly increased; Mfn2 m RNA(0.49±0.11 vs 1.15±0.30, p<0.01) decreased significantly; Bcl-2 m RNA(0.28±0.08 vs 1.13±0.25, p<0.01) decreased significantly; and Caspase3 m RNA and Bax m RNA(1.73±0.72 vs 0.52±0.11 and 0.89±0.12 vs0.49±0.06, p<0.01) increased significantly. Compared with the group of6 h, the Drp1 m RNA of liver tissue in 12 h group(0.66±0.11 vs 0.85±0.13,p<0.01) decreased significantly; Mfn2 m RNA(0.80±0.07 vs 0.49±0.11,p<0.01) significantly increased; Bcl-2 m RNA(0.33±0.11 vs 0.28±0.08,p>0.05) increased, but the difference was not statistically significant;Caspase3 m RNA and Bax m RNA(1.20±0.41 vs 1.73±0.72 and 0.51±0.20 vs 0.89±0.12, p>0.05) decreased, but the difference was not statistically significant. Compared with the group of six hours, the Drp1 m RNA of liver tissue in 24 h group(0.36±0.07 vs 0.85±0.135, p<0.01) decreased significantly; Mfn2 m RNA(1.05±0.31 vs 0.49±0.11, p< 0.01) significantly increased; Bcl-2 m RNA(0.64±0.16 vs 0.28±0.08, p<0.01) increased significantly; Caspase3 m RNA(0.21±0.13 vs 0.89 ±0.12, p<0.01)decreased significantly; but Bax m RNA(1.34±0.43 vs 1.73±0.72,p>0.05)decreased,without statistically significant.Conclusion The mitochondrial dynamics imbalanced that Drp1 expressed increase and Mfn2 decrease, which may leaded to mitochondrial dysfunction and activated mitochondrial apoptotic pathway is one of the mechanisms of ALI induced by LPS.Part2 The influence on mitochondrial dynamics and apoptosis by Mdivi-1 targeted intervention on LPS-induced ALI in miceBackground Mitochondrial dynamics was regulated by a series of fission and fusion proteins, excessive division lead to release of a large number of Caspase3 which activate mitochondrial apoptotic pathologythat mediated by Bcl-2 family and induce apoptosis and necrosis; Mdivi-1is a Drp1 specific blocker, which can block excessive mitochondrial division, effectively reduce the apoptosis and necrosis.Objective To observe the protective effect of blocking Drp1, to explore new therapeutic strategies in sepsis-liver damage.Methods 40 C57BL/6 mice were randomly divided into four groups:normal group, model group, co-solvent(DMSO) group and, 50mg/kg Mdivi-1/ DMSO intervention group, 10 in each group. The intervention group were builded the model after injected Mdivi-1/ DMSO half an hours; as the same DMSO group were builded the model after injected DMSO 0.1ml half an hour; sacrificed all the surviving mice after modeling 6 hours, tested serum ALT, AST levels; HE stained of liver tissues were analyzed; used the ELISA to assay of the ATP yield, MRCCI and Cyt-c content liver tissue and used Real-Time PCR to test the Drp1/Mfn2/Bcl-2/Bax / Caspase-3 m RNA levels of the liver tissue.Results Compared with the model group, Mdivi-1 treatment group mitochondrial ATP and MRCCI(40.07±10.96 vs 24.00±10.20 and33.28±4.79 vs 11.74±8.63, p<0.05) increased significantly, Cyt-c(2.91 ±1.32 vs 6.49±1.07, p<0.01) decreased significantly; the pathological damage index of liver(0.10±0.05 vs 0.79±0.27, p<0.05) decreased significantly;Drp1 m RNA c(1.29±0.31 vs 1.72 ± 0.42, p<0.05) decreased significantly; Mfn2 m RNA(1.51±0.38 vs 1.13 ± 0.26, p<0.01) significa-ntly increased; Bcl-2 m RNA(4.17±1.30 vs 1.86±1.15, p<0.01) increased significantly; and Caspase3 m RNA and Bax m RNA(0.62±0.14 vs 2.56±2.01 and 0.52±0.22 vs 0.99 ± 0.16, p<0.01) was significantly decline.Conclusion Mdivi-1 targeted block Drp1 that can regulate mitochondrial dynamics would be the one possible mechanism to protect the ALI in mice. |
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