Protective Effect Of Salidroside On Skeletal Muscle Atrophy In COPD Rat Caused By Smoked Cigarettes

Objective: To explore the protective effects of salidroside on skeletal muscle atrophy of COPD rat cuased by cigarette smoke, and the mechanism. To provide an experimental evidence of salidroside used in the treatment of the improvement of skeletal muscle atrophy patients with COPD.Method:Before COPD model was established, 60 male SD rats were randomly divided into six groups:control group and control+salidroside group,smoking group and smoking+salidroside group quit smoking group and quit smoking +salidroside group.The COPD model group was passive inhalated cigarette smoke 2 times a day, each time 1h, continued 4 month to establish COPD experimental model, The control group was not exposed to cigarette smoke. Continued smoking after 4 months, through the lung function was detected and pathological morphology in lung tissue was observated to evaluate the COPD model success or not. control group(n=10) and control+salidroside group(n=10), the COPD model group was redivided into stop smoking group(n=10) and stop smoking +salidroside group(n=10), smoking group(n=10) and smoking+salidroside group(n=10). The all salidroside intervented groups with salidroside(10mg/kg.bw) was used once 2 days by gastric lavage for 1 months, The all non intervented groups were used the same dose normal saline by gastric lavage for 1 months. After the intervention experiment 1 month, the body weight of rats in all groups was determined. The contents of ROS, MDA, IL-6 and TNF-α in BALF, lung tissues, blood and skeletal muscle were detected by ELISA. The protein expression of Atrogin-1 and Mu RF-1 in skeletal muscle by immunohistochemistry, the contents of Atrogin-1 and Mu RF-1 m RNA in skeletal muscle were detected by RT-PCR, the protein contents of Atrogin-1 and Mu RF-1 were detected by Western blot.Results:(1)Weight comparison: S group weighing 283.77 ± 13.13(g) compared with C group weighing 413.67 ± 15.46(g) significantly reduced, with statistical significance(p <0.05). Q group weighing 324.98 ± 15.80(g) compared with C group weighing 413.67 ± 15.46(g) significantly reduced, with statistical significance(p <0.05). Q group weighing 324.98 ± 15.80(g) compared with S group weighing 283.77 ± 13.13(g) significantly increased, with statistical significance(p <0.05). Group C group weight of 413.67 ± 15.46(g) and C/Sa group weighing 416.39 ± 13.04(g) was not statistically significant. Q/Sa group weighing 345.15 ± 10.49(g) comparing Q weight 324.98 ± 15.80(g) the increase was statistically significant(p <0.05). S/Sa group weighing 315.86 ± 24.56(g) compared with S group weighing 283.77 ± 13.13(g) the increase was statistically significant(p <0.05).(2)ROS content: ①lavage(U/ml) in C group 18.37 ± 0.19, C/Sa group 18.42 ± 0.07, Q group 54.01 ± 0.64, Q/Sa intervention group was 36.89 ± 0.71, S group 89.67 ± 1.02, S/Sa group 71.63 ± 1.70. ②lung tissue(U/mgprot) in group C 18.25 ± 0.16, C/Sa group 18.26 ± 0.18, Q group 54.10 ± 0.80, Q/Sa group 71.97 ± 1.61, S group 90.24 ± 1.00, S/Sa group 71.63 ± 1.70. skeletal m③ uscle(U/mgprot) in group C 18.20 ± 0.06, C / Sa group 18.20 ± 0.21, Q group 53.71 ± 0.76, Q/Sa group 36.75 ± 0.30, S group 88.63 ± 0.43, S/Sa group 71.36 ± 0.57. serum(U/ml) in group ④C 18.24 ± 0.19, C/Sa group 18.24 ± 0.06, Q group 53.92 ± 0.10, Q/Sa group 36.79 ± 0.27, S group 89.52 ± 0.81, S/Sa group 71.76 ± 0.37. Each specimen S group than in group C concentration, statistically significant(P <0.05). Q group than in the group C concentration, statistically significant(P <0.05). Q group concentration lower than the S group, statistically significant(P <0.05). C and C/Sa was no statistical significance. Q/Sa group lower than the Q group concentration was statistically significant(P <0.05). S/Sa group concentration lower than the S group, with statistical significance(P <0.05).(3)MDA content: ①lavage fluid(nmol/m L) in C group 9.96 ± 1.01, C/Sa group 9.75 ± 0.59, Q group 19.37 ± 1.16, Q/Sa group 15.33 ± 0.16, S group 25.29 ± 1.66, S/Sa group 21.22 ± 0.91. ②lung tissue(nmol/mgprot) in group C 9.93 ± 0.95, C/Sa group 9.90 ± 0.53, Q group 19.28 ± 1.21, Q/Sa group 15.22 ± 0.38, S group 25.22 ± 1.42, S/Sa group 21.25 ± 0.93. skeletal muscle(nmol/mgprot) in group C 9.77 ± 0.62, ③C/Sa group 9.72 ± 0.26, Q group 19.45 ± 1.24, Q/Sa group 15.20 ± 0.40, S group 25.41 ± 1.35, S/Sa group 21.27 ± 0.89. serum(nmol/m L) in ④group C 9.72 ± 0.71, C/Sa group 10.02 ± 0.51, Q group 19.84 ± 1.17, Q/Sa group 15.25 ± 0.65, S group 25.43 ± 1.41, S/Sa group 21.52 ± 1.40. Each specimen S group than in C group concentration, statistically significant(P <0.05). Q group than in the C group concentration, statistically significant(P <0.05). Q group concentration lower than that in group C, there was statistically significant(P <0.05). C and C/Sa was no statistical significance. Q/Sa set lower than the Q group concentration was statistically significant(P <0.05). S/Sa group concentration lower than the S group, with statistical significance(P <0.05).(4)IL-6(pg/ml) content: ①lavage fluid group C 13.48 ± 0.72, C/Sa group 13.68 ± 2.74, Q group 41.05 ± 4.71, Q/Sa intervention group was 25.15 ± 6.52, S group 65.58 ± 6.59, S/Sa group 49.78 ± 4.36. ②lung tissue of group C 12.62 ± 6.06, C/Sa group 12.33 ± 6.63, Q group 41.67 ± 5.25, Q/Sa group 24.38 ± 8.19, S group 63.94 ± 4.55, S/Sa group 49.48 ± 3.20. skeletal muscle group C 14.44 ± 2.17, C/Sa group 14.72 ± 7.95, ③Q group 46.50 ± 7.17, Q/Sa group 29.98 ± 5.92, S group 73.43 ± 3.89, S/Sa group 55.93 ± 6.17. ④serum group C 13.29 ± 3.26, C/Sa group 13.29 ± 2.07, Q group 39.96 ± 1.31, Q/Sa group 26.77 ± 4.42, S group 65.49 ± 7.24, S/Sa group 51.63 ± 3.92. Each specimen S group than in group C concentration, statistically significant(P <0.05). Q group than in the group C concentration, statistically significant(P <0.05). Q group concentration lower than the S group, statistically significant(P <0.05). C and C/Sa was no statistical significance. Q/Sa set lower than the Q group concentration was statistically significant(P <0.05). S group than S/Sa group lower concentration, with statistical significance(P <0.05).(5)TNF-α(pg / ml) content: ① lavage fluid group C 11.61 ± 3.70, C/Sa group 10.20 ± 3.79, Q group 39.34 ± 1.12, Q/Sa group 28.24 ± 2.83, S group 62.81 ± 1.92, S/Sa group 45.32 ± 6.09. ② lung tissue of group C 14.31 ± 4.24, C/Sa group 14.56 ± 7.55, Q group 49.14 ± 9.42, Q/Sa group 27.83 ± 1.94, S group 83.70 ± 2.35, S/Sa group 58.18 ± 2.21. ③ skeletal muscle group C 15.21 ± 1.68, C/Sa group 16.87 ± 2.94, Q group 49.79 ± 2.39, Q/Sa group 33.16 ± 9.60, S group 79.60 ± 5.58, S/Sa group 57.15 ± 5.83. ④ serum group C 15.59 ± 3.05, C/Sa group 15.46 ± 0.80, Q group 45.09 ± 3.64, Q/Sa group 30.21 ± 1.68, S group 82.80 ± 2.35, S/Sa group 59.72 ± 2.88. Each specimen S group than in group C concentration, statistically significant(P <0.05). Q group than in the group C concentration, statistically significant(P <0.05). Q group concentration lower than the S group, statistically significant(P <0.05). C and C/Sa was no statistical significance. Q/Sa set lower than the Q group concentration was statistically significant(P <0.05). S/Sa group concentration lower than the S group, with statistical significance(P <0.05).(6)Immunohistochemistry result: ①Atrogin-1 protein: Group C 150.83 ± 1.63, C/Sa group 148.78 ± 5.34, Q group 118.04 ± 3.93, Q/Sa group 128.31 ± 5.73, S group 94.32 ± 4.77, S/Sa group 108.72 ± 3.94. S Group C expression than the enhancement was statistically significant(P <0.05). Q Group C expression than the enhancement was statistically significant(P <0.05). Q group than in the S group decreased expression, statistically significant(P <0.05). Group C and C/Sa was no statistical significance. Q/Sa group than in the Q group decreased expression was statistically significant(P <0.05). S/Sa group decreased expression compared with S group, with statistical significance(P <0.05). ②Mu RF-1 protein: Group C 152.85 ± 1.25, C/Sa group 151.78 ± 4.69, Q group 120.63 ± 4.91, Q/Sa group 136.52 ± 2.74, S group 100.36 ± 5.33, S/Sa group 115.12 ± 4.05. S Group C expression than the enhancement was statistically significant(P <0.05). S group was significantly enhanced expression compared with C group(P <0.05). Q Group C expression relatively weakened statistically significant(P <0.05). Group C and C/Sa was no statistical significance. Q/Sa group than in the Q group decreased expression was statistically significant(P <0.05). S/Sa group decreased expression compared with S group, with statistical significance(P <0.05).(7)Atrogin-1 m RNA expression in multiples: Group C 0.87 ± 0.19, C/Sa group 1.06 ± 0.12, Q group, 2.72 ± 0.17, Q/Sa group, 1.67 ± 0.15, S group, 4.21 ± 0.27, S/Sa group 3.70 ± 0.20. S group than in group C increased expression was statistically significant(P <0.05). Q group than in group C increased expression was statistically significant(P <0.05). Q group S group was relatively less statistically significant(P <0.05). Group C and C/Sa was no statistical significance. Q/Sa group was compared with Q group decrease was statistically significant(P <0.05). S/Sa group was compared with S group decreased statistically significant(P <0.05).(8)Mu RF-1 m RNA expression in multiples: C group 1.11 ± 0.05, C/Sa group 1.07 ± 0.10, Q group, 2.86 ± 0.35, Q/Sa group, 1.69 ± 0.08, S group, 3.88 ± 0.33, S/Sa group 3.09 ± 0.56. S group than in group C increased expression was statistically significant(P <0.05). Q group than in group C increased expression was statistically significant(P <0.05). Q group S group was relatively less statistically significant(P <0.05). Group C and C/Sa was no statistical significance. Q/Sa group was compared with Q group decrease was statistically significant(P <0.05). S/Sa group was compared with S group decreased statistically significant(P <0.05).(9)Western blot analysis: ①Atrogin-1 protein(OD): Group C 0.15 ± 0.01, C/Sa group 0.15 ± 0.03, Q group, 0.44 ± 0.04, Q/Sa group, 0.29 ± 0.03, S group, 0.75 ± 0.06, S/Sa group 0.58 ± 0.05. S group was enhanced compared with group C, the statistically significant(P <0.05). Q group was enhanced compared with group C, the statistically significant(P <0.05). Q group was decreased compared with S group, there was statistically significant(P <0.05). Group C and C/Sa was no statistical significance. Q/Sa group was decreased compared with Q group, statistically significant(P <0.05). S/Sa group was decreased compared with S group, with statistical significance(P <0.05). ②Mu RF-1 protein(OD): Group C 0.27 ± 0.03, C/Sa group 0.28 ± 0.01, Q group, 0.76 ± 0.08, Q/Sa group, 0.55 ± 0.05, S group, 1.29 ± 0.11, S/Sa group 1.10 ± 0.10. S group was enhanced compared with group C, the statistically significant(P <0.05). Q group was enhanced compared with group C, the statistically significant(P <0.05). Q group was decreased compared with S group, there was statistically significant(P <0.05). Group C and C/Sa was no statistical significance. Q/Sa group was decreased compared with Q group, statistically significant(P <0.05). S/Sa group was decreased compared with S group, with statistical significance(P <0.05).(10) mitochondrial morphology: S group compared with C group with a large number of mitochondrial cristae,reduced mitochondrial vacuolation; mitochondria solid,the volume is reduced.Q group compared with C group with a large number of mitochondria cristae, reduced mitochondrial vacuolation; mitochondria solid, the volume is reduced.S group compared with Q group there is a lot of mitochondria cristae,reduced mitochondrial vacuolation; mitochondria solid, the volume is reduced. Group C and C/Sa group mitochondrial morphology no significant difference. Q group than Q/Sa group mitochondrial cristae,reduced mitochondrial vacuolation; mitochondria solid,increase volume reduction phenomenon.S group compared with S/Sa group mitochondrial cristae, reduced mitochondrial vacuolation; mitochondria solid, increase volume reduction phenomenon.(11)Atrogin-1, Mu RF-1 m RNA(multiple) correlation with body weight of rats: Atrogin-1, Mu RF-1 m RNA expression(times) and body weight of rats was negatively correlated(respectively: R =-0.910, P <0.01; R =-0.895, P <0.01).Conclusions:(1) Long-term smoking can cause the skeletal muscle functional disorders in COPD rats through the way of oxidative stress and inflammation mechanisms.(2) Salidroside can effectively inhibit systemic oxidative stress and inflammation,.can decrease the contents of ROS and MDA, and inhibit the levels of IL-6 and TNF-α, in BALF, blood, lung tissue and skeletal muscle of COPD rats caused by passive smoking. The results suggest that the appropriate dose of salidroside can improve skeletal muscle disorder by inhibiting the ubiquitin-proteasome channel.