| Purpose:To observe the therapeutic effect of acupuncture Zusanli treating spleen deficiency,the mechanism of acupuncture Zusanli adjusting mitochondrial oxidative stress and mitophagy of muscle cells of Spleen deficiency rats is illuminated.The mechanism of acupuncture Zusanli treating Spleen deficiency is partially confirmed and generalized by improved mitochondrial oxidative stress and mitophagy from AMPK pathway.The result will shed some lights to the treatment of Spleen deficiency with TCM therapy.Material and method:Forty-eight SPF SD male and female rats(weight 210±10g)were provided by Beijing Vital River Laboratory Animal Technology Co.,Ltd.These rats were adaptively fed in experimental animal center of Liaoning University of TCM for 1 w,which were randomly divided into normal group,spleen deficiency group,Zusanli group,and non-acupoint group,twelve rats in each group.The multi factor modeling method was used to make the model continued for 14d.The rats of spleen deficiency group,Zusanli group,and non-acupoint group were intragastric administration with Xiao Cheng Qi Tang every morning,1 time each day,and exhausted swam every afternoon,twice a day,10 minutes interval,and quantitatively fed from 8:00 am to 8:00 pm qd alt,free drinking water.The rats' weight was measured before and after the model,and the general condition of the rats was closely observed.The normal control group did not have any stimulation and normal feeding.After making the model for 14d,the rats of Zusanli group were treated by electroacupuncture Zusanli(ST 36)at 9:00 am continuous 20 min,1 time each day,and the rats of non-acupoint group were treated by electroacupuncture non-acupoint.The treatment was continued for 10d.The intensity standard of electroacupuncture is the slight tremor of the limbs of rats(Electric current:1-2mA;Dilatational wave:2Hz/100Hz;Output voltage:2V).The rats of spleen deficiency group were fixed for 20 min at the same time every day,but no electroacupuncture was used.The rats of each group after the last administration in the fasted 24h,cannot help but water.The rats were anesthetized with 10%chloral hydrate 0.6ml/100g body weight).Myocardia and musculi quadriceps femoris of rats were detected tissues.2%polyformaldehyde-2.5%glutaraldehyde fixative,experimental apparatus and so on were precooled(4?)before taking the tissues.1 × 1 ×1mm3 of myocardia and musculi quadriceps femoris were cut down with a sharp blade within 1 minute after tissue broken blood,which were fixed in the fixative,stored in 4? refrigerator.The rest of the tissues were stored in-80? refrigerator.The HPLC method for the content determination of ATP,ADP and AMP in muscle tissues of rats.The protein expression of ATP synthase,AMPK,p-AMPK,SIRT1,PGC-1?,ULK1,p-ULK1 LC3-? and LC3-? in muscle tissues of rats was detected by Western blotting.The change of ATP synthase,SIRT1 and PGC-1? mRNA was detected by fluorescence quantitative PCR method.Ultrastructure of mitochondria and mitochondrial autophagosome in muscle tissues of rats was observed by transmission electron microscope.All data was analysed by SPSS 19.0.The measured data indicated(x±s)that among the groups using single factor analysis of variance,and the comparison among groups was performed with LSD method.The differences of P<0.05 were statistically significant.Results:1.There were no significant differences with holding power of each group before making the model.After making the model for 14d,the holding power of spleen deficiency group,Zusanli group and non-acupoint group was lower than that of normal group.No differences of that had been observed between Zusanli group and non-acupoint group.The holding power of spleen deficiency group,Zusanli group and non-acupoint group was also lower than that of normal group after treating for 10d,but the holding power of Zusanli group was significantly stronger than that of spleen deficiency group2.Compared with normal group,ATP in muscle tissues of spleen deficiency group significantly reduced.ATP in skeletal muscle of Zusanli group was higher than that of spleen deficiency group.There were no significant differences with ATP in myocardia between Zusanli group and spleen deficiency group,but that of Zusanli group was a rising trend.No differences of these parameters above had been observed between spleen deficiency group and non-acupoint group.3.In contrast to normal groupAMP/ATP in muscle tissues of spleen deficiency group increased.AMP/ATP in skeletal muscle of Zusanli group was higher than that of normal group,but there were no significant differences with AMP/ATP in myocardia between Zusanli group and normal group,which was a rising trend.No differences of these parameters above had been observed on spleen deficiency group,Zusanli group and non-acupoint group.4.The protein and mRNA expression of ATP synthase in muscle tissues of spleen deficiency group was lower than that of normal group.The protein expression of ATP synthase in skeletal muscle of Zusanli group had no differences with spleen deficiency group?but the mRNA expression of ATP synthase was higher than that of spleen deficiency group.The protein expression of ATP synthase in myocardia of Zusanli group was significantly higher than that of spleen deficiency group.There were no significant differences with the protein and mRNA expression of ATP synthase in muscle tissues between Zusanli group and spleen deficiency group.In contrast to spleen deficiency group,the protein expression of non-acupoint group significantly increased,but the mRNA expression had no differences.5.There were no significant differences with p-AMPK/AMPK in muscle tissues between spleen deficiency group and normal group,but p-AMPK/AMPK of spleen deficiency group was a rising trend.Compared with normal group and spleen deficiency group,p-AMPK/AMPK in skeletal muscle of Zusanli group increased.P-AMPK/AMPK in myocardia of Zusanli group was higher than that of normal group?but no significant differences with spleen deficiency group.No differences of these parameters above had been observed between spleen deficiency group and non-acupoint group.6.The protein expression of SIRT1 in skeletal muscle of spleen deficiency group was significantly higher than that of normal group,but the mRNA expression of SIRT1 had no significant differences between spleen deficiency group and normal group.The protein expression of SIRT1 in skeletal muscle of Zusanli group was significantly higher than that of spleen deficiency group,but the mRNA expression of SIRT1 had no significant differences between spleen deficiency group and Zusanli group.There were no significant differences with the protein and mRNA expression of SIRT1 in myocardia of each group.7.Compared with normal group,the protein and mRNA expression of PGC-la in muscle tissues of spleen deficiency group significantly reduced.In contrast to spleen deficiency group,the protein and mRNA expression of PGC-la in muscle tissues of Zusanli group significantly increased.There were no significant differences with the protein and mRNA expression of PGC-la in myocardia and the protein expression of PGC-la in skeletal muscle between non-acupoint group and spleen deficiency group.The mRNA expression of PGC-la in skeletal muscle of non-acupoint group was significantly higher than that of spleen deficiency group.8.P-ULK1/ULK1 in muscle tissues of spleen deficiency group was significantly higher than that of normal group.In contrast to spleen deficiency group,p-ULKI/ULK1 in skeletal muscle of Zusanli group and non-acupoint group both significantly increased,and p-ULK1/ULK1 in myocardia of non-acupoint group decreased,and there were no significant differences with p-ULK1/ULK1 in myocardia of Zusanli group.9.Compared with normal group,LC3-?/LC3-? in myocardia of spleen deficiency group increased,and there were no significant differences with LC3-II/LC3-I in skeletal muscle of spleen deficiency group,but that in skeletal muscle of spleen deficiency group was a rising trend.LC3-II/LC3-I in skeletal muscle of Zusanli group was significantly higher than that of spleen deficiency group.LC3-?/LC3-? in myocardia of Zusanli group was significantly higher than that of normal group,but there were no significant differences with spleen deficiency group.No differences of these parameters above had been observed between spleen deficiency group and non-acupoint group.10.The mitochondrion of skeletal muscle in normal control group were evenly distributed on both sides of the Z line under transmission electron microscope,which were uniform in size just like an oval,and sharpness of border,uniform matrix,had no mitochondrial autophagy.The mitochondrion of skeletal muscle in spleen deficiency group became vacuolated,and the mitochondrion were swollen,which were not of uniform size.The mitochondrion were seriously damaged.The mitochondria damage of Zusanli group was improved,and a large number of different stages of mitochondrial autophagosome appeared.The ultrastructure of the mitochondria in non-acupoint group was similar to that of spleen deficiency group.11.The mitochondrion of myocardia in normal control group were uniform in size under transmission electron microscope,and sharpness of border,uniform matrix,had no mitochondrial autophagosome.The mitochondrion of myocardia in spleen deficiency group had unclear border,and the mitochondria were swollen and their cristae fragmented,which became vacuolated.There was a small amount of mitochondrial autophagosome.The mitochondria damage of Zusanli group was improved?and a large number of different stages of mitochondrial autophagosome appeared.The ultrastructure of the mitochondria in non-acupoint group was similar to that of spleen deficiency group.Conclusion:1.The treatment of acupuncture Zusanli could obviously improve the symptoms of spleen deficiency in rats,and it has a certain therapeutic effect on spleen deficiency.2.The mitochondrial function of the skeletal muscle of the spleen deficiency rats could be effectively improved by acupuncture Zusanli,and to a certain extent could be improved the content of the energy metabolism substance of myocardial mitochondria.3.The oxidative stress reaction of mitochondria in muscle cells of spleen deficiency rats could be effectively improved by acupuncture Zusanli.The expression of AMPK and SIRT1 was increased by acupuncture Zusanli,which activated PGC-la directly or indirectly,that could adjust mitochondrial oxidative stress of the skeletal muscle of the spleen deficiency rats.AMPK was activated by acupuncture Zusanli,which phosphorylated PGC-la directly,in order to improve mitochondrial oxidative function of rats myocardia with spleen deficiency,that maintained the normal energy metabolism of the body.The therapeutic effect mechanism of acupuncture Zusanli treating spleen deficiency was confirmed and generalized partially based on AMPK/PGC-la pathway adjusting the oxidative stress reaction of mitochondria in muscle cells of spleen deficiency rats.4.The mitophagy in muscle cells of spleen deficiency rats could be effectively improved by acupuncture Zusanli.AMPK of rats skeletal muscle with spleen deficiency was activated furtherly by acupuncture Zusanliwhich phosphorylated ULK1 directly,and ULK1/AMPK stable complex was formed,that improved mitophagy in skeletal muscle of spleen deficiency rats,and maintained the homeostasis of the internal environment in skeletal muscle cells.The therapeutic effect mechanism of acupuncture Zusanli treating spleen deficiency was confirmed and generalized partially based on AMPK/ULK1 pathway adjusting mitophagy in muscle cells of spleen deficiency rats.However,the signal pathway of adjusting mitophagy in myocardia of spleen deficiency rats will be further studied. |
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