The Effects And Mechanisms Of Zanthoxylum Alkylamides On Protein Synthesis And Catabolism In Rats

Zanthoxylum bungeanum(Z.bungeanum)is important and edible natural spices and herbal ingredient in China and some countries,its active ingredients are odor and flavor components.And,the flavor components is the numb-taste substance(Zanthoxylum alkylamides),which is important active components with unique structure(acylamides)in Z.bungeanum,antioxidant,anti-tumor,anti-inflammatory and analgesic,anti platelet aggregation and other physiological activity.Recent studies show that Zanthoxylum alkylamides can promote the oxidation of fat,reduce fat deposition and plasma triglyceride concentration in effect;in addition,through inhibition of gluconeogenesis,Zanthoxylum alkylamides also can reduce glycogen output,and repair the islet function,promote insulin secretion to improve glycolipid metabolism disorder by streptozotocin(streptozocin,STZ)in diabetic rats.Although some studies are about effects of Zanthoxylum bungeanum on the organism,there are many unresolved problems,especially,protein synthesis and catabolism.It was reported that protein synthesis and catabolism are dependent on the mTOR pathway,which means that mTOR may be as a cross site of protein synthesis and degradation,regulate protein metabolism by regulation downstream genes.However,activation of mTOR is mediated by multiple signaling pathways(nutritional factors,growth factors,energy)in upstream,and AMP-activated protein kinasev(AMP-activated protein kinase,AMPK)can negatively regulate mTOR.The ubiquitin-proteasome pathway(UPP)connected to the target proteins to make it ubiquitination through tertiary enzyme reaction,which mediates 80%-85% protein catabolism in eukaryote.However,how does effect Zanthoxylum alkylamides on the protein synthesis and catabolism,and which stimulus signal(nutritional factors,growth factors,energy)was invovled,these are unclear.Therefore,the rats were used for in vivo experiment,by qPCR and Western-blot,the effect of Zanthoxylum alkylamides on the AMPK and mTOR pathway of protein synthesis,and UPP pathway of catabolism in diabetes type 1 and healthy rats.Finally,we clarify mechanism of Zanthoxylum alkylamides on the protein synthesis and catabolism in vivo,and establish technology and method system about molecular mechanism of effects bioactive substance on protein metabolism.The main results are as follows:(1)Zanthoxylum alkylamides effect on protein metabolism in healthy SD rats32 healthy male SD rats were randomly divided into control group(Control),the groups orally treated with soybean oil dissolved Zanthoxylum alkylamides high,dose of 8mg/(kg.d)(HDG),middle dose with 4mg/(kg.d)(MDG)and low dose with 2mg/(kg.d)(LDG),and 8 rats in each group.To investigate the effects of Zanthoxylum alkylamides on protein metabolism in healthy SD rats,after feeding for 28 days,determination of physiological and biochemical indexes related to protein metabolism in the serum,liver,skeletal muscle and jejunum;the mRNA level of Myogenin(MyOG),myostatin(MSTN),?-calpain(CAPN-1)and calpastatin(CAST)were detected in skeletal muscle by qPCR.Compared with the control group,the results show that Zanthoxylum alkylamides can significantly improve skeletal muscle relative weight of healthy SD rats(p<0.05),significantly decrease the rate of abdominal fat weight,but the other organs was not significant(P>0.05);significantly increased the content of total protein,albumin and globulin in serum(p<0.05),but the effect on albumin/globulin ratio was not significant(P>0.05);significantly reduce blood urine nitrogen(BUN)content(p<0.05),but the effect on the content of Cr was not significant in serum(P>0.05);increased significantly content of triiodothyronine(T3)and insulin-like growth factor-1(IGF-I)in serum(p<0.05),but the content of thyroxine(T4),interleukin 6(IL-6)and insulin(Ins)were no significant in serum(P>0.05);the activity of ?-glutamyltransferase(?-GT)and aminopeptidase N(APN)of jejunum in middle and high dose group were significantly improved(p<0.05),but the activity of carboxypeptidase A(CPA)and dipeptidyl peptidase IV(DPP-IV)activity of jejunal were no significant(P>0.05);significantly increased the content of RNA in skeletal muscle(p<0.05),but had no significant effect on the content of DNA in skeletal muscle(P >0.05)and the high dose group and middle dose group significantly increase the ratio of RNA/DNA(p<0.05)in skeletal muscle.In addition,significantly increase mRNA levels of IGF-,MyOG,CAPN-1 and CAST in skeletal muscle(p<0.05),and significantly decreased mRNA levels of MSTN(p<0.05).The results that intragastric administration of appropriate dose of Zanthoxylum alkylamides can increase the relative skeletal muscle of healthy SD rats,reduce abdominal fat deposition significantly;significantly decreased body weight;Zanthoxylum alkylamides can change carcass composition by adjusting the energy allocation.The appropriate dose of Zanthoxylum alkylamides could significantly increase the content of total protein in serum,significantly enhance the activity of GPT in liver and promote the protein synthesis.In addition,Zanthoxylum alkylamides can promote skeletal muscle growth by down-regulation of mRNA expression of MSTN and upregulation of mRNA expression of MyOG,;skeletal muscle protein can be updated by the CAPN-1/CAST system to enhance and promote protein deposition.And the protein deposition of high dose group and middle dose group was significantly higher than that of low dose group(p<0.05).(2)The effect of Zanthoxylum alkylamides on amino acid and amino acid transporter in healthy SD ratsThe design of experiment was same with(1),To investigate whether the amino acid and amino acid transporter is a signal factor for the protein deposition in healthy SD rats,the contents of 15 kinds of amino acids in serum,liver,skeletal muscle and jejunum were determinated,and mRNA levels of the amino acid transporters in jejunum,skeletal muscle and liver were detected by qPCR.Compared with the control group,the results show that Zanthoxylum alkylamidessignificantly increased Asp,Glu,Asn,Arg,Ser and Ala content in serum and skeletal muscle of healthy SD rats(p<0.05);significantly decreased the content of Gln in serum(p<0.05);significantly increased the content of Gln in skeletal muscle and jejunum(p<0.05);increased Gln content in the liver,but not significantly(P>0.05).Significantly reduced the content of BCAA in skeletal muscle(p<0.05),the Ile content of the high dose and middle dose group significantly reduced in skeletal muscle.Zanthoxylum alkylamidessignificantly up-regulated mRNA levels of ECAA1,ASCT1,PepT1 and PAT1 in jejunum(p<0.05);significantly reduced the mRNA levels of CAT-1,y+LAT1,b0,+AT in jejunum(p<0.05);but for ATB0,and no significant effect(P>0.05).significantly increased mRNA expression of PepT1,ASCT1 and ECAA1 in liver(p<0.05),and the mRNA level of PAT1 in skeletal muscle was significantly increased(p<0.05),and the mRNA expression of ATF4,CD98 and y+LAT1 was downregulated significantly(p<0.05).The results showed that Zanthoxylum alkylamides increased glycogenic amino acid in serum and skeletal muscle,which mainly was attributed to the own synthesis of the corresponding alpha keto acid.And increased glycogenic amino acid of the high dose group and the middle dose group were significantly higher than that of low dose group in skeletal muscle(p<0.05).Based on the above results,the amino acid and amino acid transporter is not the signal factor of the effect of the Zanthoxylum alkylamideson the protein deposition in healthy SD rats.(3)The effect of Zanthoxylum alkylamides on protein synthesis and catabolism in healthy SD ratsThe design of experiment was same with(1),To further investigate the mechanism of the effect of Zanthoxylum alkylamideson the protein synthesis and catabolism in healthy SD rats.The mRNA and protein level of genes related to protein synthesis and decomposition were detected by by qPCR and Western-blot.Compared with the control group,The results show that Zanthoxylum alkylamides can significantly increased mRNA expression of IGF-I,mTOR,PI3 K and PKB in liver(P<0.05),significantly up-regulated protein level of mTOR,p-mTOR(Ser2448),PI3K(P110),PKB and p-PKB(Ser437)of liver in SD rats(P<0.05);significantly up-regulated mRNA level of IGF-I,mTOR,PI3 K,PKB,TSC2 in skeletal muscle(P<0.05),significantly increased protein level of mTOR,p-mTOR(Ser2448),PI3K(P110),PKB,p-PKB(Ser437)and TSC2 in skeletal muscle(P<0.05);the protein level of AMPK p-AMPK(Thr172)and TSC1 was not significant(P>0.05).In addition,significantly reduced the mRNA and protein levels of atrogin-1/MAFbx,MuRF1,FoxO1,Fox O3,Fox O4 and in the skeletal muscle(P<0.05).The results suggest that the effects of Zanthoxylum alkylamides on AMPK pathway was not significant in healthy SD rats(P>0.05);can increase protein synthesis by IGF-I?PI3K?PKB?mTOR pathway,and reduce protein catabolism by UPP pathway.(4)The effect and mechanism of Zanthoxylum alkylamides on protein metabolism in STZ induced diabetic rats40 male diabetic rats was used the research object,and were randomly divided into control group(Control),diabetes model group(DM),high dose(8mg/(kg.d)of intragastric group(DM-HD),the middle dose(4mg/(kg.d)group(DM-MD)and low dose(2mg/kg.d)group(DM-LD),8 rats in each group,after continuous intragastric administration for 28 days.The physiological and biochemical indexes of protein metabolism,the activity of protein metabolic enzyme and the level of hormone were determinated;and the mRNA and protein expression of genes related the protein metabolism in liver and skeletal muscle was detected by qPCR and Western-blot,to explore the effect and mechanism of Zanthoxylum alkylamides on protein metabolism in diabetic rats,and further clarify the effect of Zanthoxylum alkylamides on mechanism of protein metabolism.The results show that Zanthoxylum alkylamides can significantly reduce the content of fasting blood glucose and fructosamine of diabetes rats(P<0.05),can alleviate the organomegaly induced by diabetes,skeletal muscle weight was increased significantly(P<0.05);insulin,IGF-I,total protein,albumin and globulin/albumin ratio were significantly increased in serum(P<0.05).there are significant increases content of total protein,RNA and RNA / DNA ratio in skeletal muscle(P<0.05);can increase the mRNA expression of IGF-I,IGF-IR and InR in the liver and skeletal muscle significantly(P<0.05);The mRNA expression of PI3 K,PKB and mTOR in skeletal muscle was significantly increased(P<0.05);significantly reduced mRNA expression of atrogin-1/MAFbx,MURF1 and Fox O in skeletal muscle(P<0.05);the protein level of mTOR(total),p-mTOR(ser2448),PKB(total),p-PKB(ser437)and PI3K(P110)was significantly up-regulated in skeletal muscle(P<0.05);the protein expression of atrogin-1/MAFbx,MURF1 and FoxO in skeletal muscle was significantly decreased(P<0.05).The results showed that Zanthoxylum alkylamides can improve protein metabolism disorder induced by STZ in diabetic rats,including improvement of protein metabolism disorder in high dose group and middle dose group are significantly better than the low dose group(P<0.05).It suggested that Zanthoxylum alkylamidescan increase protein synthesis by Ins/IGF-I?PI3K?PKB?mTOR signaling pathway,and reduce protein catabolism by UPP pathway in diabetic rats.(5)The effect of Zanthoxylum alkylamides on AMPK pathway in skeletal muscle of diabetic ratsThe design of experiment was same with(4),Determination of TG(triglyceride),MDA(malondialdehyde),TC(total cholesterol)and FFA(free fatty acid)content in serum and liver of diabetic rats;In order to investigate the effects of Zanthoxylum alkylamideson AMPK pathway and its potential mechanism in skeletal muscle of diabetic rats.The mRNA and protein expression of AMPK,SIRT1,ACC,GLUT4 and other related genes were detected by qPCR and western-blot.The results showed that the content of TG,MDA and FFA in serum of diabetic rats was significantly decreased(P<0.05),and the contents of TC,TG and MDA in the liver were significantly decreased(P<0.05).The results of qPCR and Western-blot shows that Zanthoxylum alkylamides up-regulated mRNA expression of AMPK in skeletal muscle of diabetic rats(P > 0.05);but significant upregulation of AMPK(P<0.05)and p-AMPK(Thr172)protein expression,the mRNA expression of ACC(P<0.05)and protein expression of p-ACC(Ser79)and the mRNA and protein expression of GLUT4 in skeletal muscle cells membranes of diabetes rats(P<0.05).The results suggest that Zanthoxylum alkylamides can reduce the formation of fatty acid by activation of AMPK pathway and inhibition of ACC activity in skeletal muscle of diabetic rats and;at the same time,through the activation of AMPK signaling pathway mediated by GLUT4 rapid translocation,improve glucose transport rate,reduce blood glucose.Therefore,it can improve the metabolism of glucose,lipid and protein in diabetic rats by activating AMPK pathway.The increase of protein synthesis in skeletal muscle of diabetic rats was the comprehensive result of Ins/IGF-I?PI3K?PKB pathway and AMPK pathway,and Ins/IGF-I?PI3K?PKB?mTOR pathway was dominant.In summary,there are some differences in the effect and mechanism of Zanthoxylum alkylamides on protein synthesis in rats with different constitutions.In healthy SD rats,mainly increase the relative skeletal muscle weight,IGF-I content of serum and tissue,reduce abdominal fat rate,but there was no significant effect on Ins(P>0.05);promote the synthesis of the corresponding alpha keto acid;and activate mTOR the IGF-I?PI3K?PKB pathway,and the effect of amino acid/amino acid transporter signaling pathway and AMPK pathway is not significant(P>0.05).However,in diabetic rats,mainly increase skeletal muscle weight,not only significantly increased Ins content of serum(P<0.05),but increased IGF-I content of serum and tissues(P<0.05),active AMPK pathway,the increase of protein synthesis in skeletal muscle mTOR and AMPK pathway were involved,and Ins/IGF-I?PI3K?PKB pathway is dominant.Although the effects of Zanthoxylum alkylamides on the protein synthesis in different physical rats(healthy SD rats and diabetic rats)by PI3K?PKB?mTOR pathway.But there is a difference,in healthy SD rats,the signal factor of activation of PI3K?PKB?mTOR pathway is IGF-I;in diabetic rat,the signal factors are Ins and IGF.Based on previous studies,In healthy SD rats,Zanthoxylum alkylamides mainly promote protein synthesis;However,in diabetic rats,not only promote protein synthesis,but inhibit protein catabolism.According to the above results,the innovation of this paper is as follows:(1)For the first time,it was proved that protein synthesis was increased by Zanthoxylum alkylamidesthrough IGF-I ? PI3 K ? PKB ? mTOR pathway in skeletal muscle of healthy SD rats,and protein decomposition was reduced by UPP pathway.(2)The upstream pathway of mTOR,Ins/IGF-I? PI3 K ? PKB and AMPK pathway were activated by Zanthoxylum alkylamides,and protein catabolism was reduced by UPP pathway in skeletal muscle of diabetic rats.