| Background:Because TCM syndromes have cross-system pathophysiological connotations,and moreover,TCM compound formulas have complex components and the characteristics of multiple systems and multiple targets.This makes the study of syndromes or formulas from a single angle have great uncertainty.At present,exploring the modern biological basis of formula and syndrome correlation is becoming an important research hotspot in Correlation between formulas and syndromes fields."The spleen governs the muscles" is one of the important physiological properties of the spleen in TCM.Buzhong Yiqi Decoction is one of the representative formulas for invigorating the spleen qi.Clinical and experimental studies have shown that this formula has a certain effect on strengthening the spleen qi and strengthening the muscles when used for spleen deficiency and muscle weakness syndrome,but the molecular mechanism of it is still unclear.Therefore,exploring the biological basis of the relationship between Buzhong Yiqi Decoction(formula)and spleen deficiency and muscle weakness(syndrome)has important scientific significance for revealing the modern connotation of the spleen governing muscles and the molecular mechanism of Buzhong Yiqi Decoction.The clinical manifestations of sarcopenia are characterized by muscle weakness or/and atrophy,which is very close to the syndrome of spleen deficiency and muscle weakness in TCM.Studies have shown that protein metabolic imbalance is an important pathophysiological basis of sarcopenia,involving regulation disorders at different levels of system-organ-cell molecules.The previous research of our team showed that the rat model of spleen deficiency syndrome replicated by food control+exhausted swimming involves abnormal changes in multiple systems,accompanied by decreased skeletal muscle strength and pathological damage.The molecular mechanism involves energy metabolism disorders,nerve-muscles Abnormal joint function and inflammatory injury.Comparative studies of several spleen-invigorating prescriptions show that Buzhong Yiqi Decoction has a better effect on improving the muscle injury of the model,and involves the regulation of multiple pathophysiological links.Based on the above background,we speculate that the syndrome of spleen deficiency and muscle weakness in TCM is a disease characterized by inflammatory activation,abnormal energy metabolism,endocrine disorders and other links that have been explored before to participate in the disorder of muscle protein metabolism,and it has its own unique molecules.Objective:A mouse model of spleen deficiency and muscle weakness syndrome was established and evaluated by the "food control+exhausted swimming " method.On this basis,the effect on strengthening the spleen and muscles of Buzhong Yiqi Decoction was observed.And taking the imbalance of muscle protein metabolism as the starting point,in the overall,organ and molecular dimensions,explore the pathophysiological characteristics of the spleen deficiency and muscle weakness syndrome model,explore the target of Buzhong Yiqi Decoction and the biological basis of the relationship between the two.Study 1:Establishment and Evaluation of Spleen Deficiency and Muscle Weakness syndrome Model MiceMethods:KM mice were randomly divided into a control group and a model group(30 mice in each group).The model group adopted the "food control+exhausted swimming" method to establish a spleen-deficiency and muscle weakness mouse syndrome model,and the control group was fed normally.Mice in each group(1)Observe and record the appearance and behavior scores,body weight and diet of each group of mice every week.(2)Perform weight-bearing swimming exhaustion experiments on the 2nd and 4th weekends of the experiment to determine the exhaustion time of each group of mice.(3)The grip strength of mice in each group was measured with a grip strength meter on the 1th,2th,3th,and 4th weekend of the experiment.(4)The 15th and 29th days were divided into two batches,and the ELISA method was used to measure mouse serum D-xylose,GAS,MTL,VIP,IL-1?,IL-6,TNF-?.Weighing method is used to measure and calculate thymus and spleen index,muscle wet weight and muscle weight/body weight ratio.Making gastrocnemius HE slices to observe pathological changes,and use Image J software to calculate muscle fiber diameter and cross-sectional area.Results:Compared with the control group,mice in the model group,(1)The scores of activity status,skin and hair,and stool status increased significantly in the first 2-4 weeks of the experiment(P<0.01).Their weight is significantly reduced in the first 1-4 weeks(P<0.01).The food intake in the first week increased significantly(P<0.01),and the food intake in the 3-4 weeks decreased significantly(P<0.01).(2)The swimming exhaustion time was significantly shortened in the first 2-4 weeks(P<0.01 or P<0.05).(3)Grasping power is significantly reduced in the first 1-4 weeks(P<0.01),(4)In 2-4 weeks,the levels of D-xylose,GAS,MTL in serum were significantly decreased,and the levels of VIP in serum were significantly increased(P<0.01 or P<0.05).The thymus and spleen indexes were significantly reduced,the levels of IL-1? and TNF-? in serum were significantly reduced(P<0.01 or P<0.05),and IL-6 was significantly increased(P<0.01 or P<0.05).The wet weight and wet weight/body weight ratio of gastrocnemius muscle was significantly reduced(P<0.05).Pathological sections showed that the gastrocnemius muscle fibers were arranged loosely,the edges became blunt,and the gap between the muscle fibers and the muscle bundles became significantly wider.The muscle fiber diameter and cross-sectional area are significantly reduced(P<0.01 or P<0.05).Conclusions:The mouse model established by the "food control+exhausted swimming" method not only showed the appearance and behavior related to the syndrome of spleen deficiency in TCM and the changes in laboratory indicators,but also accompanied by the decline of muscle strength and muscle fiber atrophy,suggesting that the "food control+exhausted swimming" method can successfully establish spleen deficiency and muscle weakness syndrome mouse model.Study 2:Molecular mechanism of protein metabolism imbalance in muscle reduction syndrome of spleen deficiency and the intervention effect of Buzhong Yiqi DecoctionMethod:KM mice were randomly divided into control group,model group,Buzhong Yiqi Decoction high-dose group and low-dose group(15 mice in each group).Except for the control group,the rest of the groups used the "food control+exhausted swimming " method to establish spleen-deficiency muscles Weak mouse model,for 4 weeks,the high and low dose groups were given Buzhongyiqi granules aqueous solution at 3.8 g/kg·d and 1.9 g/kg·d respectively from the 15th day.Mice in each group,(1)Observe appearance behavior scores,weight,diet,swimming exhaustion time,and grip strength according to the method of Study 1.(2)On the 29th days of the experiment,the levels of CRH and GnRH in hypothalamus and D-Xylose,GAS,MTL,VIP,IL-1?,IL-6,TNF-?,ACTH,CORT,GH,and T in serum of each group was determined by ELISA.The thymus and spleen index,the wet weight and muscle weight/body weight ratio,pathological changes,muscle fiber diameter and cross-sectional area were determined according to the method of Study one.The levels of GR?,IGF-1,IGF-1R,INS,INSR,GH,T,IL-1?,IL-6,TNF-?,myostatin and ACTR2B in the gastrocnemius muscle of each group of mice were determined by ELISA.The relative protein expression of pi3k,p-akt,p-mTOR,p-s6k1,p-eIF4B,IRS1,p-Smad3 and p-Foxo3a in the gastrocnemius muscle of each group of mice were determined by Western Blot.Immunohistochemical method was used to detect the positive expression of Atrogin-1 and MuRF-1 in the gastrocnemius muscle.Results:Compared with the control group,mice in the model group,(1)The score of activity status increased significantly in the 1-4 weeks of the experiment(p<0.05),and the scores of activity status,skin and hair,and stool status increased significantly in the 2-4 weeks of the experiment(P<0.01).The food intake in the first week was significantly increased(P<0.01),and significantly reduced in the 3-4 weeks(P<0.01).The body weight and exhaustion time in the first 1-4 weeks Significantly shortened(P<0.05).(2)The levels of D-xylose,GAS,MTL in serum were significantly reduced,and the level of VIP in serum was significantly increased(P<0.01 or P<0.05).The thymus and spleen indexes were significantly reduced(P<0.01).Gastrocnemius wet weight and muscle weight/body weight ratio were significantly reduced(P<0.01 or P<0.05).Electron microscopy showed that the gastrocnemius muscle fibers are arranged loosely,the edges become blunt,and the gap between muscle fibers and muscle bundles is significantly wider.Both the diameter and cross-sectional area of muscle fibers were significantly reduced(P<0.01).Grasping power decreased significantly from the 2nd and 4th week(P<0.01).The total protein of gastrocnemius muscle was significantly reduced(P<0.05).The levels of INS,INSR,IGF-1 and IGF-1R in the gastrocnemius muscle were significantly decreased(P<0.01),the relative protein expressions of pi3k,p-akt,mTOR,p-s6kl and p-eIF4B in the gastrocnemius muscle were significantly reduced(P<0.05),the levels of Myostatin,ACTR2B and the positive expression of Atrogin-1 and MuRF-1 were significantly increased(P<0.01 or P<0.05),the relative protein expressions of p-Smad3 and p-Foxo3a were significantly increased(P<0.01 or P<0.05).The levels of CRH in hypothalamic and ACTH,CORT,IL-6 in serum and GR-?,IL-1?,IL-6,TNF-? in the gastrocnemius muscle were significantly increased(P<0.01 or P<0.05),the levels of GnRH in hypothalamic,GH,T,IL-1?,TNF-? in serum and GH,T in gastrocnemius were significantly reduced(P<0.01 or P<0.05).Compared with the model group,mice in the high and low dose groups,(1)The scores of activity status,skin and hair,and stool status were significantly reduced in the 3-4 weeks of the experiment(P<0.01 or P<0.05).The body weight increased significantly in the 3-4 weeks(P<0.01).The food intake increased significantly in the 4th week(P<0.01).The exhaustion time was significantly prolonged in the 4th week(P<0.05).(2)The level of D-xylose in the serum of the high-dose group was significantly increased,and the level of VIP in the serum was significantly reduced(P<0.01 or P<0.05),and the levels of serum GAS and MTL in the high-and low-dose groups were significantly increased(P<0.01 or P<0.05).The thymus and spleen indexes were significantly increased(P<0.01 or P<0.05).The morphology of muscle fibers in the high-dose group was similar to that in the control group through electron microscopy.The arrangement of muscle fibers in the low-dose group was slightly loose and full,and the gap between muscle fibers and muscle bundles was slightly widened.The diameter and cross-sectional area of muscle fibers increased significantly in the high and low dose groups(P<0.01 or P<0.05).In the 4th week,the grasping power was significantly increased(P<0.01).The total gastrocnemius protein in the low-dose group was significantly increased(P<0.05).The levels of INS,INSR,IGF-1 and IGF-1R in the gastrocnemius muscle were significantly increased(P<0.01 or P<0.05),and the levels of Myostatin and ACTR2B were significantly reduced(P<0.05).The positive expression of Atrogin-1 and MuRF-1 was significantly reduced(P<0.01 or P<0.05).The relative protein expression of pi3k,p-akt and mTOR all increased significantly(P<0.01 or P<0.05),and the relative protein expression of p-s6kl in the high-dose group increased significantly(P<0.05).The levels of CRH in hypothalamic,ACTH,CORT,IL-6 in serum and GR-?,IL-1?,IL-6,TNF-? in gastrocnemius were significantly reduced(P<0.01 or P<0.05)and the levels of GnRH in Hypothalamus,T,L-1?,TNF-? in serum and GH,T in gastrocnemius were significantly increased(P<0.01 or P<0.05)of the high and low dose groups.The level of GH in serum was significantly increased in the high-dose group(P<0.05).Conclusions:The muscle atrophy mechanism of this model mouse involves different levels of the whole,organs,tissues,and molecules.On the overall level,the excessive activation of HPA axis function promotes protein degradation,while the reduction in the secretion of hormones related to the endocrine system is not conducive to protein synthesis.At the organ tissue level,the high expression of inflammatory factors in muscle tissue further promotes protein degradation.On the molecular level,the imbalance of muscle protein metabolism involves the imbalance between the mTOR/INS and UPS/myostatin signaling pathways.Buzhong Yiqi Decoction has obvious effects on strengthening the spleen qi and strengthening the muscles of mice with spleen deficiency and muscle weakness syndrome.Its mechanism of action involves the regulation of the neuro-endocrine-immune multi-system,the metabolic balance of muscle protein,and the regulation of mTOR/INS and UPS/myostatin signaling pathways.The above research results firstly provide a certain pharmacological basis for the clinical application of Buzhong Yiqi Decoction to invigorate the spleen qi and strengthen muscles.Secondly,it is of great significance to reveal the modern connotation of the spleen governing muscles in TCM,the molecular mechanism of the effect on strengthening the spleen qi and muscles of Buyiqi Decoction,and the biological basis of Buzhong Yiqi Decoction-spleen deficiency and muscle weakness syndrome. |
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