Objective:
To investigate the effects of Ligustrazine on denervated skeletal muscle cellapoptosis and expression of bcl-2, bax effect, looking for delaying atrophy of denervated skeletalmuscle in new ways.Methods 54 SPF grade healthy Wistar rats, 3-4 weeks of age, body mass (200±10) g, eithermale or female, were randomly divided into 9 groups: normal control group, respectively (A ),loss of neural control group ( B, C, D, E ), and the intervention group (F, G, H, I ).
Application of10% chloral hydrate (2ml/kg) intraperitoneal injection anesthesia, rat femoral posterior incisionof right lower limb, exposure of the right sciatic nerve, the normal control group (0d ) only exposed the sciatic nerve does not cut off; denervated control group, the intervention ofLigustrazine on sciatic nerve cut more than the 10mm, proximal flip 180 degrees, with the surrounding tissue suture, the establishment of right lower limb denervated gastrocnemius animalmodel.
After surgical intervention:
The intervention of Ligustrazine on intraperitoneal injection ofLigustrazine Injection 80mg/kg·d; denervated controls peritoneal injection dose of normal saline.
After zeroth, 2, 7, 14, 28 days are a group of rats, weighing body weight after death, and bothsides of the gastrocnemius muscle and muscle wet weight, the refrigerator at minus 80 degrees Celsius, standby.
Using RT-PCR and Western Blot detection in each period of Bcl-2 andbaxmRNA and protein expression level. The use of deoxyribonucleotide transferase mediatednick end labeling ( TUNEL ) method for the detection of muscle cell apoptosis. Using SPSS13.0statistical software to undertake statistical processing.
Results
This study confirmed the rat gastrocnemius muscle after denervation of muscle wetweight ratio is decreased,28d is about 32% in the control group.The intervention of Ligustrazinegroup 2d on muscle wet weight ratio has no significant effect(P>0.05);postoperative7d,14d,28dand the intervention group compared with the control group,although the reduction of musclewet weight ratio (28d approximately for normal control group64%), but were significantly higherthan control group(P<0.05).
Rat gastrocnemius muscle after denervation bcl-2mRNA at each timepoint is reduced continuously, while baxmRNA increased continuously, but were higher thanthose of normal control group. And denervation in comparison to the control group, theexpression of bcl-2mRNA relative content is low, but were higher than the same period ofdenervated control group (P<0.05); on the contrary, and denervation in comparison to the controlgroup, the intervention of Ligustrazine group baxmRNA while increased, but were lower thanthe same period of denervation in control group(P<0.05).
Rat gastrocnemius muscle afterdenervation of the protein expression of bcl-2 at each time point is reduced continuously,and the protein expression of Bax at each time point gradually increased,but were higher than those ofnormal control group. And denervation in comparison to the control group,the intervention ofLigustrazine group bcl-2 protein expression is low, but were higher than the same period ofdenervated control group(P<0.05);while the Bax protein expression is increased, but were lowerthan the same period of denervation in control group (P<0.05).
The denervated group apoptosisrate gradually increased, and compared with normal controls the difference was statisticallysignificant(P<0.05), by treating,2d apoptosis rate was significantly lower than the same period inthe denervated group(P<0.05), then with the denervated time prolonged, its level of expressionwhile increasing trend, but but always lower than with the same period in denervated controls expression levels (P<0.05).
Correlation analysis showed, in the denervated group in baxexpression and muscle wet weight ratio were negative correlation(r=-0.776，P<0.05), and theapoptosis rate was positively related to relationship(r=0.811，P<0.05); denervated group in bcl-2expression and muscle wet weight positive correlation between the ratio of (r=0.829，P<0.05 ),and the apoptosis rate was negative correlation (r=-0.790，P<0.05 ), indicate that bcl-2 and baxexpression and apoptosis of muscle cells, rate of muscle wet weight ratio; and bax and bcl-2expression were negative linear correlation relationship(r=-0.821，P＜0.05),indicates thedenervated gastrocnemius atrophy and the decrease of bcl-2 expression and bax expression wasrelated to the increased.
Conclusion
Ligustrazine may help prevent denervated skeletal muscle atrophy through theinhibition bcl-2 or bax expression