Effects Of Acute Exercise And Acupuncture Treatment On Skeletal Muscle Extracellular Matrix

1. ObjectivesThe purpose of this project is to investigate skeletal muscle ultrastructure, the content changes of different type of collagens, the balance of MMPs/TIMPs in rat skeletal muscle extracellular matrix after acute eccentric exercise, analyse the relationship among MMP2, MMP9, TIMP1 and TIMP2, reveal the mechanism of acupuncture effect on exercise-induced skeletal muscle damage and remodeling extracellular matrix of skeletal muscle.2. Materials and MethodsEight-week-old Sprague-Dawley rats (n=96, body wt 220±6.9g), randomly assigned to 4 groups which named sedentary control(C), acupuncture control (CA), exercise group (E) and acupunctured exercise group (EA), were used in this study. The animals were adapted to the living room for 3 days and made to run on a motor-driven treadmill with-16°downhill tracks at a speed of 16m/min for total 90 min. Acupuncture control and acupunctured exercise rats were intervened after exercise. The animals were placed under anesthesia and killed by drawing blood from aorta abdominalis. The tissues were collected from vastus intermedius muscle of the experimental rats immediately,24 hours,48 hours,72 hours,120 hours after exercise. All the samples were stored in-80°fridge later. Transmission electron microscope(TEM) was used to observe the skeletal muscle ultrastructure, Scanning electron microscope(SEM) was used to analyse the morphological changes of collagen fibers, Western blotting and RT-PCR were adopted to detect gene and protein expression of typeⅠ,Ⅲ,Ⅳcollagen, MMP2, MMP9, TIMP1 and TIMP2 in this research.3. Results3.1 Acute eccentric exercise could facilitate increasing of collagen fibers, however acupuncture could inhibit it. SEM results revealed that collagen hyperplasia came up in skeletal muscle of E group rats, not EA rats,120 hours post exercise. mRNA level of type I and III collagen of E group rats were up regulated to maximal level 24 hours after exercise comparing with immediate killed rats, P<0.05, whereas mRNA level of typeⅣcollagen in E group was low than C group and significantly decreased comparing with immediate killed rats, P<0.05. The mRNA level of type I and III collagen in EA rats significantly increased 48 hours after exercise comparing with immediate killed rats, however, mRNA level of typeⅣcollagen in EA rats was highest 24 hours after exercise comparing immediate killed rats, P<0.05.3.2 Both MMP2 and MMP9 mRNA level in E group rats significantly increased comparing with sedentary control rats 24 hours after exercise, P<0.05. At the same time, the protein expression of MMP2 and MMP9 of E group rats were up to maximal level, but the protein expression of MMP2 had a clear change comparing with immediate killed rats, P<0.05, MMP9 did not. Comparing with C group rats, acupuncture significantly up-regulated the mRNA level of MMP2 and MMP9 48 hours after exercise, P<0.05. Whereas the protein level of MMP2 and MMP9 in the skeletal muscle of EA rats increased to the top respectively 24hours,72 hours after exercise comparing with C group rats, P<0.05.3.3 Twenty four hours after exercise, mRNA level of TIMP1 and TIMP2 in skeletal muscle of exercise rats were highest and changed significantly comparing with sedentary control rats, P<0.05. There were no clear changes of TIMP1 and TIMP2 protein expression, although both of them were slightly elevated after exercise. In skeletal muscle of acupunctured exercise rats, mRNA level of TIMP1 and TIMP2 increased respectively 24 hours and 48 hours after exercise to the maximal level comparing with sedentary control rats, P<0.05. The protein content of TIMP1 in acupunctured exercise rats was elevated dramaticly 72 hours after exercise comparing immediate killed rats, P<0.05, whereas TIMP2 protein level of this group rats increased immediately to the highest level after exercise, declined gradually to the lowest level 120 hours after exercise comparing with immediate killed rats, P<0.05.4. Conclusion 4.1 Acute eccentric exercise could stimulate collagens increasing in rat skeletal muscle. Acupuncture altered collagens expression to facilitate the recovery and regeneration of injuried skeletal muscle.4.2 Acupuncture induced the collagen degradation in order to facilitate the recovery and regeneration of injuried skeletal muscle via increasing the expression of MM2 and MMP9.4.3 TIMP1 and TIMP2 played different roles in inflammation of injuried skeletal muscle, it seemd that TIMP1 was more important in the early phase of injuried skeletal muscle recovery.