The Expression And Effect Of NF-κB And Matrix Metalloproteinase 2,9 In Chronic Alcoholic Myopathy

Chronic alcoholic myopathy is a kind of myopathy because of long time alcohol misuse. It has been reported that there are about 40%-60%alcoholist whose muscle was affected. Their frequently clinical appearance were having difficulties in gait, various muscle symptoms such as cramps, local pain and reduced muscle mass. So, chronic alcoholic myopathy affects the quality of patients'life severely. However, there is merely few clinical and experimental reports about CAM in our coutry, even fewer than alcoholic liver disease (ALD) and alcoholic myocardiopathy. Lots of researchers have observed that nuclear factor-κB (NF-κB) and matrix metalloproteinase 2,9 (MMP-2, MMP-9) play a key role in skeletal muscle weakness and amyotrophy. We experimentalize in order to investigate these factors whether produce a marked effect on the pathogenthy of alcoholic myopathy or not. By doing this, we may find some available method and it is important for the prevention and therapy of chornic alcohol myopathy.Object: The project is on the basis of chronic alcoholic myopathy model of rat, to dynamically observe the change of NF-κB and MMP-2, MMP-9 in muscle cell, observe the function in the mechanism of alcohol myopathy, move forward a single step to explain the pathogenesy of this disease from gene and protein leve. We also provide a helpful platform for other metabolic myopathy.Methods:(1) 80 health, male, Sprague-Dawley rats(weight 180~200g), were chosen to sep up the CAM rat myopathy model ,to observe the rat bodyweight, the change of praxiology and the patho-change of muscle tissue. (2) Operated group was treated with alcohol. Control group was given NS which contain the same volume to ethanol. At the same time, they are feeding the same improved food with high fats and sufficient nutrients. At the end of 12 and 18 weeks, rats were killed, taken the muscle of posterior limb, after separating the gastroenemius,soleus,plantaris, to prepare the frozen section of the each kind of muscle respectively to be confirmed to form the myopathy model by the histochemical stain. (3) RT-PCR,Western blot and immunohisto-chemistry stain were respectively used to detect the expression of NF-κB,MMP-2 and MMP-9 during different period. Gelatin zymography was used to detect the activation and secretion of MMP-2 and MMP-9 in gastroenemius,soleus,plantaris during early,intermediate and final stage of experimental group. Picric- sirius red polarized light was used to detect the accrementition and reconstitution of mesenchymal fibers in the muscle specimens from the operated group and normal controls.Results:(1) After being given alcohol, the pathological lesion of theⅡ-type fibers is more severe than theⅠ-type fibers, the treated rat show up the typical patho-change, such as the myofibrosis,necrosis, the shapes of the muscle fiber atrophy become triangle,strip or irregular, phlegmasia cell smear and muscle fiber regenerate,the connective tissue among the muscle tissue become hyperplasia, and so on.(2) RT-PCR method shows that along with the alcohol taking,the expression of NF-κB and MMP-9 increase gradually, especially in gastroenemius and plantaris.The expression of MMP-2 has no significant difference between operated group and normal controls. But it increase notablely after stioping treating.(3) Immunohistochemistry stain shows: A lot of positive express of NF-κB were found in the cytoblast and endochylema of operated group plantaris and the activity express of iNOS wasn't found in control.(4) Western blot method shows that the expression of NF-κB and MMP-9 increase gradually on protein level. Ethanol can activate and promote the expression of NF-κB. As the same with the result of RT-PCR, MMP-2 increases notablely after stioping treating.(5) The result of Picric- sirius red polarized light method shows thatthe accrementition and reconstitution of mesenchymal fibers were markedly in experimental group compared with control group, especially in plantaris.Ⅰ-type collagen dominates in mesenchymal, IOD ratio ofⅠ/Ⅲchanged.(6) Gelatin zymography method shows that the expression of pro-MMP-9,middle-MMP-9 and active-MMP-9 in gastroenemius and plantaris of operated group increase notablely,MMP-2 increases notablely after stioping treating.Conclusions(1) The up-regulation of the expression of nuclear factor-κB was closely correlation with the genesis of CAM, which suggest that NF-κB may play an important role in inflammatory reaction and proteolysis in CAM.(2) The up-regulation of the expression of MMP-9 perhaps takes part in the accrementition and reconstitution of mesenchymal fibers (Ⅰ-type collagen dominates in mesenchymal, IOD ratio ofⅠ/Ⅲchanged). MMP-9 can facilitate inflammatory cell infiltration, aggravate the development of CAM.(3) The expression of MMP-2 were correlation with the revival of CAM.