| 【Objective】To verify the effect of electroacupuncture stimulation of Zusanli(ST36)and Yanglingquan(GB34)on sepsis-induced muscle atrophy by negatively regulating autophagy and further explore its related mechanisms.【Methods】(1)Sprague-Dawley(SD)rats of SPF grade were randomly divided into 4groups: Sham group(Sham)(n = 6),Sepsis group day 3(Sepsis 3 d)(n = 5),Sepsis group day 7(Sepsis 7 d)(n = 5)and Sepsis group day 14(Sepsis 14 d)(n = 5).Sepsis model was established in SD rats by ligating and perforating caecum.In the sepsis group,50% of the caecum was ligated and perforated.Gastrocnemius muscle,tibialis anterior muscle and diaphragm muscle were extracted on the 3rd day,7th day and14 th day.The gastrocnemius muscle fibers were stained with hematoxylin and eosin;the changes of muscle fibers at different time points were observed under the microscope;the changes of muscle fiber diameter were measured;the expression of LC3 B in gastrocnemius muscle was detected by immunohistochemical technique;Western blotting was used to detect the expression of LC3 B,p62 and Beclin-1 in gastrocnemius muscle,tibialis anterior muscle and diaphragm muscle,and to determine the appropriate time of electroacupuncture intervention.(2)SD rats of SPF grade were randomly divided into 7 groups: Sham group(Sham)(n = 5),Sepsis group(Sepsis)(n = 6),Electroacupuncture groups(EA)which were divided into 2 Hz group(EA 2Hz)(n = 6),5 Hz group(EA 5Hz)(n = 6),20 Hz group(EA 20Hz)(n = 6),50 Hz group(EA 50Hz)(n = 6).SD rats sepsis model was established as above,and the abdomen was only opened and closed in the sham model group;the rats in the electroacupuncture group were stimulated at Zusanli(ST36)and Yanglingquan(GB34).The gastrocnemius muscle,tibialis anterior muscle and diaphragm muscle were extracted after appropriate days of electroacupuncture intervention.The changes of body weight of rats were recorded;the muscle tissues were stained with hematoxylin and eosin;the changes of muscle fiber diameter were observed under the microscope,and screening the best parameters of electroacupuncture.Western blotting was used to detect the protein expression of autophagy markers LC3 B,p62 and Beclin-1,and to screen the optimal parameters of electroacupuncture.(3)Select the optimal parameter group of electroacupuncture and the gastrocnemius muscle tissues of the corresponding sham group and sepsis group,and detect the expression of various autophagy-related genes LC3 B,ULK-1,Beclin-1,ATG13,ATG14,ATG12,ATG5 and ATG16 L in muscle fibers by q RT-PCR method;The expression of autophagy classical markers LC3 B,p62 and autophagy-related proteins ULK-1,p-ULK-1,Beclin-1,p-Beclin-1 and pathway proteins AKT,p-AKT,m TOR,p-m TOR were detected by Western blotting.【Results】(1)After cecal ligation and puncture,the gastrocnemius muscle in Sepsis group had abnormal morphology and significantly decreased muscle fiber diameter compared with Sham group on the same day.The muscle fiber diameter decreased gradually with time in Sepsis group;The expression of LC3 B in the gastrocnemius muscle of Sepsis group was significantly increased in the 3rd day group and in the 7th day group,and decreased in the 14 th day group compared with Sham group;The expression of autophagy markers in gastrocnemius muscle,tibialis anterior muscle and diaphragm muscle detected by Western blotting showed that compared with Sham group,the ratio of LC3 B II/I and the expression of Beclin-1 increased and the expression of p62 decreased in Sepsis group at 3 and 7 days,the increase degree in 3days group was greater than that in 7 days group,and the change was the smallest in14 days group;Considering that the change of autophagy level was the greatest in 3days,in order to facilitate the observation of the trend,the appreciate electroacupuncture stimulation was determined to be 7 days,On the 3rd day,part of the rats’ muscles were sampled,and the rest of the rats were treated with electroacupuncture for 7 days,and only the changes of body weight were recorded.(2)After caecal ligation and puncture,the changes of body weight recorded 7days after electroacupuncture intervention showed that the body weight of the Sham group increased slightly,and the Sepsis group decreased gradually compared with the Sham group,with the largest increase on the 3rd day after operation;Compared with Sepsis group,EA groups were higher than Sepsis group at 3 days after operation(except 20 Hz group),and EA groups were higher than Sepsis group at 7 days after operation.The results showed that compared with Sham group,the muscle fiber diameters in the Sepsis group and EA group were decreased,and the muscle fiber diameters in the EA group were higher than those in the Sepsis group.The optimum frequency of EA was 10 Hz for gastrocnemius and diaphragm,and 20 Hz for tibialis anterior.The expression of autophagy markers detected by Western blotting showed that the ratio of LC3 B II/I and the expression of Beclin-1 were increased and the expression of p62 was decreased in Sepsis group compared with Sham group;Compared with Sepsis group,the level of autophagy was decreased in EA groups,and the appreciate stimulation frequency of autophagy inhibition was 10 Hz in gastrocnemius and diaphragm,and 20 Hz in tibialis anterior.The results were consistent with the electroacupuncture parameters screened by muscle fiber diameter.(3)The gastrocnemius muscle of Sham group,Sepsis group and EA 10 Hz group was extracted,and the expression of autophagy-related genes was detected by q RT-PCR.The expression of LC3 B,ULK-1,Beclin-1,ATG13,ATG14,ATG12,ATG5 and ATG16 L m RNA in Sepsis group was higher than that in Sepsis group.Compared with Sepsis group,the expression of autophagy-related genes in EA group was lower.The expression of autophagic classic marker proteins and related proteins detected by Western blotting showed that the expression of LC3 B II and LC3 B II/I ratio increased,while the expression of LC3 B I and p62 decreased in Sepsis group compared with Sham group.The expressions of ULK-1,p-ULK-1,Beclin-1and p-Beclin-1 were increased in EA group.Compared with Sepsis group,the expressions of autophagy classical marker proteins and related proteins were decreased in EA group.(4)Compared with Sham group,the expression of p-AKT and p-m TOR in Sepsis group decreased;compared with Sepsis group,the expression of p-AKT and p-m TOR in EA group increased after EA intervention.【Conclusions】(1)Sepsis induces skeletal muscle atrophy,and the degree of atrophy is related to the level of autophagy.(2)Electroacupuncture alleviated sepsis-induced skeletal muscle atrophy,and the sensitivity of gastrocnemius,tibialis anterior and diaphragm to different frequencies of electroacupuncture stimulation was different.(3)Electroacupuncture can negatively regulate the level of skeletal muscle autophagy by activating the autophagy pathway AKT-m TOR,and alleviate sepsis-induced skeletal muscle atrophy. |
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