| Objective:To investigate the effect of Tanshinone on the CK.. LDH activity in rats'serums and the histology and ultrastructure of gastrocnemius muscles and the apoptosis-related factors, such as caspase-3,8,9,after exhaustive exercise.This study was designed to explore the probable mechanism of Tanshinone on rats'gastrocnemius muscles apoptosis after exhaustive exercise.Methods:All male SD rats, weighing 180-220g, were randomly divided into two groups including quiet control group(A) and exercises groups(B),exercises groups were assigned to the following groups:exhaustive exercises group(BO),exercises+low-dose Tanshinone(B1),exercises+middle-dose Tanshinone(B2),exercises+high-dose Tanshinone(B3),exercises+Vitamin E(B4)(8 rats in each group).No treatment was given to quiet control group,common breed. Exercises groups had taken the exhausting movement. Among them, B1?B2?B3?B4 group were given an intraperitoneal injection of 3mg/kg/d of Tanshinone,6mg/kg/d of Tanshinone,12 mg/kg/d of Tanshinone and 25 mg/kg/d of Vitamin E,respectively.All groups took blood serum and gastrocnemius muscles to freeze and preserve after 5 weeks. Observe the change of morphous structure of gastrocnemius muscles tissues by HE stainning light microscope and transmission electron microscope,and detect the change of creatine kinase (CK) and lactatdehydrogenase (LDH) of serum,The immunohistochemical techniques were used to detect the expression of Caspase-3,8,9 protein.While,apoptotic cells and the apoptotic index were checked by TUNEL methods. Real-time PCR was applied to identity the expression of Caspase-3 mRNA.Results:1 the comparing results of CK?LDH of serum1.1 The activity of CK?LDH of serum in the normal control group were 996.57±64.23% 618.17±72.96,respectively;Comparing to the normal group,the activity of CK and LDH of serum in exhaustive exercise group were 1961.60±305.74 (P<0.05)?1398.20±95.62 (P< 0.01),respectively,significantly increasing.1.2 Comparing with the exhaustive exercise group, the activity of CK of serum in low-dose,moderate-dose,high-dose Tanshinone group were 1334.20±139.56?1188.80±91.93? 1337.20±130.91 (P<0.05),cutting down obviously.While, the level of LDH of serum were1113.30±159.58 (P<0.05)?802.00±120.34 (P<0.01)?1094.80±147.17 (P<0.05), cutting down significantly.1.3 Comparing with the exhaustive exercise group, the activity of CK and LDH of serum in Vitamin E group were 1226.20±159.73 (P<0.05).1079.40±85.83 (P<0.01), decreasing significantly.2 Morphological observation(1)HE stainning showed that in model group the gastrocnemius muscle fibers became swelling and arranged irregularly and these structures were disorganized;The transversus striation was not obvious. part of sarcolemma were incomplete;The gastrocnemius muscle nuclear had an uneven distribution with cell nucleus accumulated or part of the nucleolus disappeared;Muscle gap disappeared.While the Tanshinone group can reduce the injury significantly,Most of gastrocnemius muscle fibers arranged regularly and organized clearly,only a few of that became swelling;Cell nucleus of muscle distributed equally under sarcolemma without proliferation, swelling, karyopyknosis;Interspace of muscle fibers was normal.(2)The transmission electron microscope results showed that in nomal group the gastrocnemius muscle fibres arranged normally and clearly;and the construction of mitochondria were normally distributued between the myofibrils.While in model group the gastrocnemius muscle fibres were disordered alignment and fuzzy,and the sarcomere were disordered and Z line was abnormal with fuzzy,rheology,distorted.the mitochondria was vacuolar degeneration, swelling,disslution.However,comparing with the model group,the gastrocnemius muscle fibres injury in different dose tanshinone were reduced,that the obvious result was in the medium dose group.3.The results of immunohistochemical testThe results showed that the Caspase-3?8?9 cell staining in model group was significantly deeper than nomal group,and the immunohistochemical score was obviously higher than nomal group (P<0.01);While the Caspase-3?8?9 cell staining in Tanshinone group and Vitamin E group were both lighter than model group, and the immunohistochemical score was obviously lower than model group (P<0.01 or P<0.05)4 The results of TUNEL testThe numbers of the apoptotic cells in model group increased obviously, which were higher than that in normal group (P<0.01); the numbers of the apoptotic cells in Tanshinone group were lower significantly than that in model group (P<0.01); the numbers of the apoptotic cells in Vitamin E group were lower significantly than that in model group (P< 0.05)5 The results of Real-time PCRThe expression of Caspase-3 mRNA in model group is higher significantly than that in normal group (2-??Ct> 2); There was no significant difference between the model group and the low dose Tanshinone group (0.5< 2-??Ct< 2);While,the expression level of Caspase-3 mRNA in middle dose and high dose of Tanshinone group was lower than that in model group (2-??Ct< 0.5); and the expression level of Caspase-3 mRNA in Vitamin E group is lower than that in model group (2-??Ct< 0.5)Conclusion:1. Different lose of Tanshinone can reduced the activity of CK and LDH, thus had some protective effect on the rats muscle injury in exhausting exercise.The different dose of Tanshinone did not have the dose-dependent protective effect on the exercise muscle injury, Moderate dosage Tanshinone,compared with other dosages,had a better protective action2. Different lose of Tanshinone can improve the pathological morphology of gastrocnemius muscle,lessen the damage structure of gastrocnemius muscle and alleviate its injury,which group had the most obvious effect was the middle dose Tanshinone group.3. The Tanshinone can adjust the change of apoptosis-related factors Caspase-3,8,9 protein and Caspase-3mRNA, and lead to the amelioration of damage status of gastrocnemius muscle. |
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