| ObjectiveIn order to research the preventive and therapeutic effect and its mechanisms of buyang huanwu decoction on denervated tibial muscle atrophy of rats, this project aims to observe the morphology and functions of the injuried nerve and its target muscle by using the crushed common peroneal nerve as a model, and to explore the molecular mechanisms of Buyang Huanwu Decoction delaying denervation muscle atrophy by the GeneChip analysis. We hope that this study will contribute to the better prevention and cure denervated muscle atrophy by traditional Chinese medicine.Method1. Collect literature about flaccidity zheng systematically, investigate the etiology,pathogenesis,therapeutic principles and method from the qi-blood and zang-fu zheng differentiation. Assemble documents of the buyang huanwu decoction on the compose,compatibility,pharmacologic actions and clinical application on the peripheral nerve lesion and denervated muscle atrophy.2. The Experimental Study on the preventive and therapeutic effect of buyang huanwu decoction on denervated tibial muscle atrophy of ratsAfter 60 male Sprague-Dawley rats were subjected to Common Peroneal nerve crush model of 5 mm injury, they were randomly divided into 6 groups for daily intragastric administration of drugs:BYHWD high-dose(25.92 g/kg), medium-dose (12.96 g/kg), low-dose groups(6.48 g /kg), mecobalamin group (625μg/kg, positive control), model group, sham operation group. Normal saline was for model group and the sham operation group. The drug administration lasted for 18 days. Footprint test was performed at the 18th days after surgery to evaluate the toe spread function (TSF); Electrophysiology was also performed to determine the amplitude of compound muscle action potentials (CMAPs) and nerve conductive velocity(NCV). At the same time, the number of regenerated myelinated nerve fibers, thickness of myelin sheath and cross sectional area and wet weight ratio of tibial muscle were also evaluated by histomorphology. All these were to observe the regeneration and functional rehabilitation of crushed Common Peroneal nerve and the preventive and therapeutic effects of buyang huanwu decoction on denervated tibial muscle atrophy of rats.3. The study on the molecule mechanism of buyang huanwu decoction delaying denervated tibial muscle atrophy of rats After 20 male Sprague-Dawley rats were subjected to Common Peroneal nerve crush model of 5 mm injury, they were randomly divided into 2 groups for daily intragastric administration of drugs:BYHWD high-dose(25.92 g/kg) and model group. The method of model construction and administering drug were the same as that of experiment 1. At the7th,18th days after surgery, the GeneChip analysis were performed. At the same time, qRT-PCR was also used to validate some differential expression gene. All this is to explore the possible molecule mechanism of buyang huanwu decoction delaying denervated tibial muscle atrophy of rats.Results1. The denervated muscle atrophy belong to "flaccidity zheng". Ii is asthenia in origin and asthenia in superficiality. The main pathomechanism is qi and blood deficiency and stagnation of vessel of blood. The main therapeutic principle is reinforcing qi and activating blood circulation. Buyang huanwu decoction is the correct prescriptions.2. TSF:all groups increased gradually after operation. At the 12th day after operation, the TSF in BYHWD high-dose group (P<0.01) and in mecobalamin group(P<0.05) increased significantly compared with that of model group. At the 15th and 18th day after the operation, the TSF in BYHWD high-dose group,medium-dose group and mecobalamin group also increased significantly compared with that of model group. (P<0.05 or P<0.01).3. The proximum and distally amplitude of CMAP:Compared with the sham operation group, model group,BYHWD groups and mecobalamin group all descended obviously(P<0.01): compared with the model group, BYHWD high-dose,medium-dose groups and mecobalamin group heightened significantly (P<0.01)4. NCV:Compared with the sham operation group, the NCV of model group,BYHWD groups and mecobalamin group all descended obviously(P<0.01); the NCV in BYHWD medium-dose group (P<0.05),high-dose group (P<0.01) and mecobalamin group (P<0.01) were also increased significantly compared with that of model group.5. The section area of tibial muscle in the sham operation group was comparatively large, morphous was rule, connective tissue was little; compared with the sham operation group, the section area in the model group reduced, morphous was not rule, fiber structure was chaos, intercellular space widened significantly, connective tissue hyperplasia was obviously, muscle fiber atropy was severity; compared with the sham operation group, the section area in the BYHWD group and mecobalamin group muscle fiber atropy was slightly; connective tissue hyperplasia was not obviously.6. The wet weight ratio:Compared with the sham operation group, the wet weight ratio of model group,BYHWD groups and mecobalamin group all descended obviously (P<0.01) compared with the model group, the wet weight ratio in BYHWD medium-dose group(P<0.05), high-dose group (P<0.01) and mecobalamin group (P<0.01) were also increased significantly.7. The section area of tibial muscle:Compared with the sham operation group, the section area of model group,BYHWD groups and mecobalamin group all descended obviously(P<0.01); the section area in BYHWD group and mecobalamin group all increased significantly compared with that of model group (P<0.01); there was also striking variation between model group and mecobalamin group (P<0.05)8. At the 18th day after operation, by cholinesterase dyeing in the sham operation group were Sepia,oval or round; the BYHWD high-dose group and mecobalamin group has little chage; the quantity of motor end plates in the BYHWD medium-dose group,low-dose group and the model group had lessen, shape was not rule gradually, the edge were rough; coloring were slight gradually.9. NF fluorescence histochemistry dyeing showed that:the neuritis in the sham operation group were dense and in good order; the neurites in the model group were raritas and chaos; the BYHWD high-dose group and the mecobalamin group had more neurite, and the BYHWD medium-dose group and low-dose group had lessen neurite.10. Electron microscope showed that nerve fibers in the sham operation group were mature,thick,dense; the nerve fibers in the BYHWD group and the mecobalamin group were comparatively mature,thick,dense; the nerve fibers in the model group were sparseness,chaos,thin and immature.11. The number of regenerated myelinated nerve fibers and thickness of myelin sheath Compared with the sham operation group, the model group,BYHWD groups and mecobalamin group all descended obviously (P<.01); BYHWD high-dose,medium-dose group and mecobalamin group were all increased significantly compared with the model group (P<0.01).12. Genechip:At the 7th day after operation, four genes had up-regulation expression of mRNA, three genes had down-regulation expression of mRNA; at the 18th day after operation, sixteen genes had up-regulation expression of mRNA, thirteen genes had down-regulation expression of mRNA. In the differential expression genes, Angptl4 had the most obviously change which were correlated with the blood neogenesis; the next was Pik3c2g which can activate AKt and had general biological effects. The biological functions of these differential expression gene can be divided into two type:the one can postpone denervated muscle atropy directly which promote the synthesis of protein and inhibit the decompose of protein, the other can delay denervated muscle atropy indirectly. The first functions are as foiiows:synthesis of protein,cell cycle,ani-apoptosis,polyubiquitinated pathway,cell proliferation and differentiation. The second functions are as foiiows:blood remodeling,blood neogenesis,improving microcirculati on,neuroprotection,collagen synthesis,energy synthesis,anti-oxidant damage. The sort by the frequency of occurrence were as foiiows:energy synthesis(8),blood neogenesis(5,ani-apoptosis(5),anti-oxidant damage(4),neuroprotection(4). Otherwise, some gene has obscure function and need lucubrate. The mRNA expression of Angptl4 and Pik3c2g were validated by the qRT-PCR, the results were the same as that of genechips. So, the gengchip analysis were reliable.13. The results of qRT-PCR were the same as the Genechip, so, the conclusions of the Genechip were right.Conclusion1. BYHWD has a significant effect of prevention and therapy on denervated tibial muscle atrophy of rats. At the same time, it can accelerate the axon growth and myelinogenesis,promote nerve regeneration and functional rehabilitation which indirectly delay the denervated tibiai muscle atrophy of rats.2. The preventive and therapeutic effect of buyang huanwu decoction on denervated tibial muscle atrophy of rats are relevant with the regulation of many genes. The mechanism may be as follows:promoting energy synthesis and blood neogenesis,ani-apoptosis,anti-oxidant damage and neuroprotection, we will need the further research.3. The PI3K/Akt signal transduction pathway can accelerate synthesis of protein and inhibit decompose of protein,promote blood neogenesis and the hyperplasia and differentiation of muscle satellite cells. So, the PI3K/Akt signal transduction pathway probably play an important role in delaying the denervated muscle atropy which is one of the mechanisms. |
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