肾衰营养胶囊改善CRF大鼠骨骼肌萎缩的作用及其机制研究
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摘要
一、目的：
营养不良是慢性肾衰竭(Chronic renal failure, CRF)患者最常见的并发症之一,其不仅具有明显的蛋白储备降低、血白蛋白水平下降、贫血等营养不良的特征,而且更突出地表现为进行性骨骼肌萎缩,瘦体质量丢失,机体蛋白质储备减少,并与患者不良预后密切有关。CRF营养不良病因及发病机制十分复杂。CRF患者摄食减少,尿毒症毒素引起的恶心、呕吐,酸中毒,以及腹透和血透丢失血浆蛋白质和氨基酸等各种原因均可导致营养不良。随着近年来对蛋白质代谢的深入研究,逐步认识到蛋白分解增加和蛋白合成减少在CRF营养不良中起主导作用。研究发现,泛素蛋白酶体系统(Ubiquitin proteasome system, UPS)参与了蛋白质及能量异常代谢,数据显示细胞内80%～90%的蛋白通过UPS途径降解。UPS在体内蛋白降解过程中发挥主要作用,两条肌肉特异性的E3连接酶：肌萎缩Fboxl (Muscle atrophy F-box, MAFbx,也称作Atrogin-1)和MurF-1(肌环指蛋白-1),在肌萎缩时显著激活。近年来,细胞凋亡在骨骼肌萎缩中的重要作用已经受到重视。文献报道,骼肌萎缩与肌细胞核的数量减少有关,而肌细胞核的数量主要是通过凋亡途径丢失的,骨骼肌萎缩与肌纤维凋亡有关。骨骼肌萎缩状态下,肌纤维促凋亡蛋白Bax、caspases的表达增加与抗凋亡蛋白Bcl-2的表达下调,说明线粒体途经的凋亡在分解代谢状态下发挥重要作用,并且其作用发生在蛋白降解之前。最新研究报道,Wnt7a还可以通过其受体Fzd7,激活蛋白激酶B/哺乳动物雷帕霉素靶蛋白(AKT/mTOR)信号通路,促进肌管肥大和肌纤维增粗。在给予杜氏进行性肌营养不良症(Duchenne muscular dystrophy,DMD)模型小鼠注射Wnt7a蛋白后,其肌纤维明显增粗,证实Wnt7a能促进肌肉组织生长和修复。虽然国内外大量研究文献都表明UPS活化、肌细胞凋亡增加及细胞内Wnt7a/Akt信号通路受损是导致骨骼肌萎缩的主要原因,但它们在CRF骨骼肌中的表达及作用尚缺乏研究,有必要探讨其在CRF骨骼肌萎缩中的作用及机制。CRF营养不良属于中医“关格”、“虚劳”等范畴。CRF营养不良多见面色萎黄,神疲乏力,纳差,消瘦,舌质淡,苔厚腻诸症。从祖国医学角度则认为与脾胃功能虚弱密切相关。盖脾为后天之本,气血生化之源,饮食入胃后,通过脾之运化,将精微物质化生气血,使之充养脏腑经络、四肢百骸、筋骨皮脉,即“中焦受气取汁变化而赤,是谓血”,亦谓“生化之源,则在于脾胃”。CRF时脾胃功能虚弱,水谷精微不化气血而成水湿浊毒。据此我们认为CRF营养不良其病在肾,关键在脾,脾胃功能失调贯彻CRF营养不良全过程。脾胃运化功能恢复,则后天补先天,有利于肾元恢复,气血生化有源,则营养状况得以改善。治疗中,鉴于CRF病机为本虚标实,虚实夹杂,当攻补兼施,正邪兼顾,治以补气血,益脾肾,泄湿浊,使补泄熔于一炉,扶正不留邪,祛邪不伤正。在此法指导下我们选用“人参养荣汤”加泻浊之品治疗CRF营养不良的患者,结果该方可以增加病人的食欲,提高血浆白蛋白,改善营养,提高患者的生活质量。肾衰营养胶囊是在“人参养荣汤”的基础上加减化裁并经科学的生产工艺研制而成,具有健脾补肾,益气养血,通腑泻浊之功效,在临床CRF营养不良的预防和治疗中取得客观疗效。本课题通过观察肾衰营养胶囊对CRF营养不良模型大鼠肾功能、肾脏病理改变、低蛋白血症、贫血以及骨骼肌萎缩的影响,检测大鼠骨骼肌UPS、细胞凋亡及Wnt7a/Akt信号通路的表达,初步探讨肾衰营养胶囊改善CRF营养不良及骨骼肌萎缩的机制,为肾衰营养胶囊防治CRF营养不良及骨骼肌萎缩,提供客观的理论实验依据。
二、方法：
取雄性SD大鼠,以5/6肾切除的方法制作CRF模型,将CRF的大鼠随机分为CRF营养不良模型组(Model)、肾衰营养胶囊组(SSYYJN)、和开同组(α-酮酸,KA)三组各10只。三组大鼠分别给予生理盐水、肾衰营养胶囊8g·kg-1·d-1、开同灌胃1g·kg-1·d-1,同时三组均继续给予低蛋白(4%酪蛋白)饲料喂养。另设假手术组(Control)10只,以生理盐水灌胃,正常蛋白饲料喂养。干预16周后,取大鼠血、尿进行血清肌酐(苦味酸法)、血清尿素氮(尿素酶法)、血清白蛋白(溴甲酚绿法)、Hb(碱化Hb比色法)、红细胞计数(血球计数仪)、血清转铁蛋白受体(免疫散射比浊法)、血清促红细胞生成素水平(多功能酶标仪)和24h尿蛋白(磺柳酸-硫酸钠比浊法)检测；留取肾脏组织包埋切片,进行Masson染色和电镜检测,观察肾脏纤维化的变化；获取胫骨前肌包埋切片,行HE染色测定肌纤维横截面积的变化；通过离体培养肌肉,检测单位时间内掺入的14+C-苯丙氨酸的放射性含量,分析蛋白合成代谢的变化；通过骨骼肌进行离体有氧培养,检测培养液中酪氨酸释放率,分析蛋白分解代谢的变化；免疫组化检测骨骼肌组织Ub蛋白的表达；免疫荧光检测骨骼肌组织MAFbx、MuRF-1蛋白；采用TUNEL法检测骨骼肌细胞凋亡的变化；Real-time PCR (RT-PCR)检测骨骼肌组织MAFbx、MuRF-1、FoxO1mRNA的表达；蛋白质印迹法(Western Blot)检测骨骼肌组织Wnt7a、phospho-Akt、phospho-mTOR、phospho-p70S6K、Bax、Bcl-2、 Cleaved caspase-3和GAPDH蛋白的表达；实验数据采用SPSS13.0统计软件进行分析。
三、结果：
1. SSYYJN对大鼠体重、胫骨前肌(Tibialis anterior, TA)湿重、干重及干重体重比的影响
术前各组大鼠体重无统计学差异(P>0.05)。体重在Model组显著下降(P<0.01),予以SSYYJN和KA干预可使其体重增加(P<0.01),且SSYYJN组较KA组增加更为显著(P<0.05)；TA湿重、干重在Model组显著下降(P<0.01),予以SSYYJN和KA干预可使其质量增加(P<0.01),但SSYYJN组和KA组间无显著性差异((P>0.05)；TA干重体重比在Model组显著减少(P<0.01),予以SSYYJN和KA干预可使其比值增加(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)；TA横截面积在Model组显著减少(P<0.01),予以SSYYJN和KA干预可使其大小增加(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)。
2. SSYYJN对大鼠血白蛋白、贫血相关指标的影响
血清白蛋白在Mode1组显著下降(P<0.01),予以SSYYJN和KA干预可使其水平增加(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)；血红蛋白(Hb)、红细胞(RBC)在Model组显著下降(P<0.01),予以SSYYJN和KA干预可使其水平增加(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)；血清转铁蛋白受体在Model组显著下降(P<0.01),予以SSYYJN和KA干预可使其水平增加(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)；血清促红细胞生成素在Model组显著下降(P<0.01),予以SSYYJN和KA干预可使其水平增加(P<0.01),且SSYYJN组较KA组增加更为显著(P<0.05)。
3. SSYYJN对大鼠肾功能指标的影响
术前各组血尿素氮、血肌酐无统计学差异。血尿素氮在Model组、SSYYJN和KA组显著下降(P<0.01),且KA组下降更为显著(P<0.01)；血肌酐在Model组显著升高(P<0.01),予以SSYYJN和KA干预可使其水平下降(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)。尿蛋白排泄率在Model组显著升高(P<0.01),且随时间进展而逐渐增加,予以SSYYJN和KA干预可使其水平下降(P<0.01)。
4. SSYYJN对大鼠肾脏病理的影响
Masson染色显示,Control组肾脏未见明显组织学异常,Model组则病变显著,出现明显肾小球硬化,小管间质病变更为显著,表现为小管扩张、萎缩、基底膜增厚,严重间质纤维化和炎症细胞浸润。给予SSYYJNIKA干预使GSI和TIS下降显著(P均0.05)。电镜结果显示,Control组肾脏无明显组织学异常,Model组病变显著,肾小球上皮细胞肿胀,线粒体空泡变性,胞浆脱落,细胞核裸露,内皮细胞核染色质边集,部分足突融合,系膜基质明显增生。近曲小管部分微绒毛脱落,线粒体肿胀,基质增生,染色质边集,上皮细胞脱落。远曲小管上皮细胞呈碎片状脱落,形成凋亡小体。SSYYJN改善肾脏病变：小球上皮细胞肿胀明显减轻,线粒体电子密度均匀,无胞浆和足突脱落,近曲小管微绒毛、线粒体膜结构清晰。远曲小管间质增生较轻,无胞浆脱落。
5. SSYYJN对大鼠骨骼肌蛋白合成和降解代谢的影响
通过对大鼠骨骼肌组织的4+C-苯丙氨酸释放率和酪氨酸掺入率检测发现,酪氨酸掺入率在Model组骨骼肌显著下降(P<0.01),提示Model组的蛋白合成代谢下降,予以SSYYJN和KA干预可使其掺入率增加(P<0.01),且KA组较SSYYJN组下降更为显著(P<0.01)；14+C-苯丙氨酸释放率在Model组显著增加(P<0.01),提示Model组的蛋白分解代谢加剧,予以SSYYJN和KA干预可使其释放率下降(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)。
6. SSYYJN对大鼠骨骼肌UPS、FoxO1mRNA表达的影响
首先通过对大鼠骨骼肌组织的免疫组化检测发现,Ub蛋白的AOD值在Model组显著增加(P<0.01),予以SSYYJN和KA干预可使其表达下降(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)；然后通过对大鼠骨骼肌组织的免疫荧光和Real-time PCR检测发现,MAFbx的平均荧光强度(MFI)、mRNA在Model组显著增加(P<0.01),予以SSYYJN和KA干预可使其表达下降(P<0.01),且KA组较SSYYJN组下降更为显著(P<0.01); MuRF-1MFI、mRNA在Model组显著增加(P<0.01),予以SSYYJN和KA干预可使其表达下降(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05); FoxO1mRNA在Model组显著增加(P<0.01),予以SSYYJN和KA干预可使其表达下降(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)。
7. SSYYJN对大鼠骨骼肌凋亡信号通路的影响
首先通过对大鼠骨骼肌组织的TUNEL检测发现,骨骼肌细胞凋亡率在Model组显著增加(P<0.01),予以SSYYJN和KA干预可使其凋亡率下降(P<0.01),且KA组较SSYYJN组下降更为显著(P<0.05)。然后通过对大鼠骨骼肌组织的Western Blot检测发现,Cleaved caspase-3蛋白在Model组显著增加(P<0.01),予以SSYYJN和KA干预可使其表达下降(P<0.01),且KA组较SSYYJN组下降更为显著(P<0.05); Bax蛋白在Model组显著增加(P<0.01),予以SSYYJN和KA干预可使其表达下降(P<0.01),且KA组较SSYYJN组下降更为显著(P<0.05)；Bcl-2蛋白在Model组显著减少(P<0.01),予以SSYYJN和KA干预可使其表达增加(P<0.01),且SSYYJN组较KA组增加更为显著(P<0.05)。
8. SSYYJN对大鼠骨骼肌Wnt7a-Akt/mTOR信号通路的影响
Western Blot检测发现,Wnt7a蛋白在Model组显著减少(P<0.01),予以SSYYJN和KA干预可使其表达增加(P<0.01),且SSYYJN组较KA组增加更为显著(P<0.05); Frizzled-7蛋白在Model组显著减少(P<0.01),予以SSYYJN和KA干预可使其表达增加(P<0.01或P<0.05),但SSYYJN组与KA组间差异无统计学意义(P>0.05); p-Akt蛋白在Model组显著减少(P<0.01),予以SSYYJN和KA干预可使其表达增加(P<0.01),且KA组较SSYYJN组增加更为显著(P<0.05)；p-mTOR蛋白在Model组显著下降(P<0.01),予以SSYYJN和KA干预可使其表达增加(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05); p-p70S6K蛋白在Model组显著下降(P<0.01),予以SSYYJN和KA干预可使其表达增加(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)。
四、结论：
1.CRF营养不良模型大鼠存在低白蛋白血症、贫血、骨骼肌萎缩等营养不良问题。SSYYJN可增加血清白蛋白、血红蛋白、红细胞计数、血清转铁蛋白受体、血清促红细胞生成素水平,改善骨骼肌萎缩。
2.CRF骨骼肌萎缩与UPS激活有关,该通路的激活可导致骨骼肌分解代谢旺盛,肌萎缩关键基因(MAFbx and MuRF1)表达升高,可能是CRF肌肉萎缩的机制之一。
3.CRF骨骼肌萎缩与细胞凋亡有关,促凋亡相关基因Bax的表达增加及抑凋亡相关基因Bcl-2的表达减少在骨骼肌细胞凋亡的发生机制中起着重要作用。
4.CRF骨骼肌萎缩与Wnt7a-Akt信号通路的下调有关,该通路的下调使Akt/mTOR和Akt/FoxO1信号途径异常,导致骨骼肌合成代谢下降和分解代谢增加,可能是CRF肌肉萎缩的另一个机制。
5. SSYYJN可以改善CRF骨骼肌萎缩,它可能是通过以下几个途径实现的：(1)抑制CRF骨骼肌UPS激活；(2)抑制CRF骨骼肌细胞凋亡,其机制可能是通过抑制促凋亡基因Bax的表达和增加抑凋亡基因Bcl-2的表达有关；(3)上调CRF骨骼肌Wnt7a/Akt信号通路。
Objective:
Malnutrition is one of the most common complications in chronic renal failure (CRF) patients, which not only has significant decreased storage protein, low serum albumin levels and anemia, but also highlighted the performance of progressive skeletal muscle atrophy, loss of lean body mass, decreased body storage protein, associated with poor prognosis in CRF patients. The etiology and pathogenesis of malnutrition with CRF is extremely complex. Reduced food intake, uremic toxin-induced nausea, vomiting, acidosis, and loss of plasma proteins and amino in peritoneal dialysis and hemodialysis patients could lead to malnutrition. In recent years, a number of studies have focused on protein metabolism, which contributed to our better understanding increase in protein degradation and decrease in protein synthesis, which played an important role in the pathogenesis of malnutrition with CRF. Studies have found that the ubiquitin proteasome system (UPS) involved in the abnormal protein and energy metabolism, showing80%-90%protein in cells were degraded by the UPS. UPS play an important role in the degradation of muscle proteins, two muscle-specific E3ligases:Fboxl muscular atrophy (MAFbx, also known as Atrogin-1), and Muscle-ring finger protein-1(MurF-1), were significantly increaed when muscle atrophy. In recent years, the apoptosis in the role of skeletal muscle atrophy has been focused on. Studies have found that the apoptosis decreased number of nucleus, which were related with muscle atrophy. The increased in pro-apoptotic Bax, caspases protein and the decreased in Bcl-2protein induced apoptosis in a catabolic state. It reported that Wnt7a binding to Fzd7directly activated the Akt/mTOR growth pathway, thereby inducing myofibre hypertrophy in differentiated myofibres. Duchenne muscular dystrophy (DMD) in mice after injecting Wnt7a protein, significantly enlarged its muscle fibers, which confirmed Wnt7a can promote muscle atrophy. Although studies suggested that activation of UPS, increase in muscle cell apoptosis and downregulation of Wnt7a-Akt/mTOR signaling pathways were the main reason which leading to muscle atrophy, but the expression of them in skeletal muscle of CRF has been unknown, it is necessary to explore its role in the muscle atrophy with CRF. Capsule of Shengshuai Yingyang (SSYYJN) was empirical prescription based on "Renshen Yangrong tang", malnutrition with CRF patients were treated with SSYYJN for many years. Our pre-clinical observation showed that SSYYJN has a better clinical curative effect on malnutrition of CRF patients. To profoundly study the role of UPS, apoptosis and Wnt7a-AKT/mTOR signaling pathwany in the mechanism of skeletal muscle atrophy, and to explore the role of Wnt7a in the pathogenesis of in CRF with malnutrition and the molecular mechanism of SSYYJN for treatment of CRF with malnutrition.
Methods:
Male rats were randomly assigned to either the5/6nephrectomy group or the sham-operated group. Those5/6nephrectomy rats were randomized into CRF model group, KA group, and SSYYJN group. The three group were fed with4%casein. The sham operated group as control group. The KA group and SSYYJN group were treated with Ketosteril and capsule of Shenshuai Yingyang for16weeks. The general status of rats, body weight was observed during the experiment. During and after treatment, the blood parameters such as ALB, BUN, Scr,24hUpro, Hb, PA, Tf, and EPO were detected, and the pathological change of renal tissue were stained by Masson and electron microscopy. The size of tibia anterior (TA) muscle was measured under HE staining and calculated with software. Protein synthesis was measured in vitro in the soleus muscle using the incorporation of14+C-phenylalanine. Protein degradation was measured using tyrosine release from the isolated muscle. The expression of Ub protein was measured by immunohistochemistry. The expression of MAFbx, MuRF-1was measured by immunofluorescence. DNA strand breaks were assessed by fluorescent labelling of terminal dUTP nick end labelling (TUNEL) using the onestep TUNEL apoptosis assay kit, TUNEL-positive cells were counted, and then their densities were calculated. The mRNA expression of MAFbx, MuRF-1and FoxO1were measured by RT-PCR. Expression of Wnt7a、phospho-Akt、 phospho-mTOR、phospho-p70S6K、Bax、Bc1-2、Cleaved caspase-3and GAPDH were detected by Western Blot.
Results:
1. The effects of SS YYJN treatment on body weight and TA muscle weight
The body weight of each group has no significant differences before the intervention. After the intervention, the body weight of Model group was significantly heavier than control group. The body weights of SSYYJN and KA groups were substantially higher than the Model Group (P<0.01), and the body weight of SSYYJN was higher than the KA Group (P<0.05). The wet and dry weights of TA muscle from Model group was a significant reduction (P<0.01) when compared with the Control Group. SSYYJN group and KA groups were significantly higher ((P<0.01) than the Model group, but no statistical differences were observed between the between SSYYJN and KA groups (P>0.05). The dry weight of TA muscle corrected for body weight from Model group was a significant reduction (P<0.01) when compared with the Control Group. SSYYJN group and KA groups were significantly higher ((P<0.01) than the Model group, but no statistical differences were observed between the between SSYYJN and KA groups (P>0.05). The mean cross-sectional area of TA muscle from Model group was a significant reduction (P<0.01) when compared with the Control Group. SSYYJN group and KA groups were significantly higher ((P<0.01) than the Model group, but no statistical differences were observed between the between SSYYJN and KA groups (P>0.05).
2. The effects of SSYYJN treatment on serum albumin and Anemia-related indicators.
Results showed that the level of serum albumin from Model Group was significantly lower than the Control group (P<0.01), the level of serum albumin from SSYYJN and KA groups were higher than the Model group, but no statistical differences were observed between the between SSYYJN and KA groups. Model Group Hb and RBC count is significantly lower than control group (P<0.01), SSYYJN and KA Hb and RBC count higher than the Model group, SSYYJN group Hb and RBC count higher than the KA Group (P<0.01). Serum transferrin receptor was lower in the Model groups than in the Control group (P<0.01), the level of serum transferrin receptor from SSYYJN and KA groups were higher than the Model group, but no statistical differences were observed between the between SSYYJN and KA groups. Serum Erythropoietin was lower in the Model groups than in the Control group (P<0.01), the level of Erythropoietin from SSYYJN groups was higher than the Model and KA groups (P<0.01).
3. The effects of SSYYJN treatment on proteinuria and renal function
Blood biochemical values of each group before the intervention has no significant difference. Intervention the values of blood urea nitrogen in the Model group was significantly lower than Control group (P<0.01), the values of blood urea nitrogen from SSYYJN and KA groups were lower than the Model group. The level of serum creatinine from Model Group was significantly higher than the Control group (P<0.01), the level of serum albumin from SSYYJN and KA groups were higher than the Model group, but no statistical differences were observed between the between SSYYJN and KA groups. The levels of urinary albumin excretion was highest in the Model group, and increased progressively with time, show progressive deterioration of renal function, and SSYYJN reduced the urinary protein.
4. The effects of SSYYJN treatment on renal tissue damage
Masson stain showed that no histologic abnormalities of the kidneys were observed in the control group. In the5/6nephrectomy groups, glomerulosclerosis and interstitial fibrosis progressed, especially in the Model group (P<0.01). Compared with the Model group, the SSYYJN and KA group showed more decrease in the glomerulosclerosis index and interstitial fibrosis score (P<0.01), but no statistical differences were observed between the between SSYYJN and KA groups (P>0.05).
5. The effects of SSYYJN treatment on muscle protein synthesis and protein degradation.
The rate of protein synthesis was significantly lower in the Model group than that in the Control group (PO.01), and SSYYJN and KA supplementation increased the rate of protein synthesis when compared with the Model group (P<0.01). The rates of protein degradation were significantly higher in the Model groups than in the Control group (P<0.01), and the rates of protein degradation from SSYYJN and KA groups were lower than the Model group, but no statistical differences were observed between the between SSYYJN and KA (P>0.05).
6. The effects of SSYYJN treatment on UPS
The protein content of ubiquitin was examined by immunohistochemical analysis. Marked immune positive staining of ubiquitin was found to be localised in the cytoplasmic region of scattered muscle fibres in the muscle of the5/6nephrectomy groups, especially in the Model group (P<0.01). The percentage of ubiquitin-positive areas calculated through semi-quantitative analysis was found to be significantly larger in the SSYYJN and KA groups than in the Model groups (P<0.01), but no statistical differences were observed between the between SSYYJN and KA (P>0.05). The expression of MAFbx and MuRF1conducive to skeletal muscle atrophy was examined by immunofluorescent staining. The mean fluorescence intensity of MAFbx and MuRF1remarkably increased in the Model group than that in the Control group (P<0.01), while the intensity was tamed with SSYYJN and KA treatment when compared with the Model group and SSYYJN group was higher than KA group. The mRNA expression of MAFbx and MuRF1assessed by RT-PCR showed significant up-regulation in the Model group but down-regulation with KA and SSYYJN intervention and SSYYJN group was higher than KA group, which is consistent with the results of immunofluorescence studies of MAFbx and MuRF1. The mRNA expression of FoxO1assessed by RT-PCR showed significant up-regulation in the Model group but down-regulation with KA and SSYYJN intervention but no statistical differences were observed between the between SSYYJN and KA groups.
7. The effects of SSYYJN treatment on apoptosis signaling pathway
DNA fragments and the level of cleaved caspase-3in skeletal muscle were evaluated by TUNEL staining and immunoblotting. TUNEL-positive nuclei of TA muscle was observed with significant increases in the Model group as compared with the Control groups (p<0.01), but distinct decrease in the SSYYJN and KA group as compared with the Model group, distinct decrease in the KA group as compared with the SSYYJN group. The semi-quantification of cleaved caspase-3also showed a similar result with TUNEL staining. Furthermore, to determine whether Bcl-2family proteins would regulate caspase-3dependent apoptotic pathway, additional immunoblotting was performed to examine the expression of Bax and Bcl-2protein. Findings demonstrated upregulated level of Bax but downregulated level of Bcl-2in the Model group as compared with the Control group (p<0.01), while upregulated level of Bcl-2but downregulated level of Bax with SSYYJN and KA treatment as compared with the Model group (p<0.01).
8. The effects of SSYYJN treatment on the Wnt7a/Akt/p70S6K signaling pathway
Evidence has been provided indicating that Wnt7a directly activated the Akt/p70S6K pathway to induce myofibre hypertrophy. We examined Wnt7a, p-Akt, p-mTOR and p-p70S6K protein levels by immunoblotting. The data indicate a lower level of Wnt7a protein in the Model group than that in the Control group (p<0.01), whereas treatment with SSYYJN and KA increased the level of Wnt7a protein (p<0.01), SSYYJN increased the level of Wnt7a protein were significantly higher than KA. Meanwhile, downward trend in Akt, mTOR and p70S6K phosphorylation in the Model group was also observed compared with the Control groups (P<0.01), but SSYYJN and KA supplementation prompted an increase as compared with the Model group (P<0.01). SSYYJN increased the level of p-Akt protein than KA, no statistical differences in the protein level of p-mTOR and p-p70S6K were between the SSYYJN and KA groups.
Conclusion:
1. SSYYJN can improve malnutrition in CRF by increasing the level of blood serum albumin, hemoglobin, red blood cell count, transferrin receptor and serum erythropoietin.
2. The increases in MAFbx and MuRF1expression are linked to the proposed role of the UPS associated with muscle atrophy in CRF.
3. Apoptosis is closely related to muscle atrophy in CRF. The increase of Bax gene expression and decrease of Bcl-2gene expression induced apoptosis in skeletal muscle.
4. Down-regulation of the Wnt7a/Akt signaling pathway is closely related to muscle atrophy in CRF, which leading to abnormal of Akt/mTOR and Akt/FoxO1signaling pathway were correlated with lower rates of protein synthesis and higher rates of protein degradation.
5. SSYYJN can effectively improve muscle atrophy in CRF. The mechanism may be as follows:(1) SSYYJN can prevent the activation of the UPS in CRF skeletal muscle;(2) SSYYJN can inhibit muscular cell apoptosis in CRF skeletal muscle;(3) SSYYJN can reverse declining expression of Wnt7a/Akt signaling pathway in CRF skeletal muscle.
营养不良是慢性肾衰竭(Chronic renal failure, CRF)患者最常见的并发症之一,其不仅具有明显的蛋白储备降低、血白蛋白水平下降、贫血等营养不良的特征,而且更突出地表现为进行性骨骼肌萎缩,瘦体质量丢失,机体蛋白质储备减少,并与患者不良预后密切有关。CRF营养不良病因及发病机制十分复杂。CRF患者摄食减少,尿毒症毒素引起的恶心、呕吐,酸中毒,以及腹透和血透丢失血浆蛋白质和氨基酸等各种原因均可导致营养不良。随着近年来对蛋白质代谢的深入研究,逐步认识到蛋白分解增加和蛋白合成减少在CRF营养不良中起主导作用。研究发现,泛素蛋白酶体系统(Ubiquitin proteasome system, UPS)参与了蛋白质及能量异常代谢,数据显示细胞内80%～90%的蛋白通过UPS途径降解。UPS在体内蛋白降解过程中发挥主要作用,两条肌肉特异性的E3连接酶：肌萎缩Fboxl (Muscle atrophy F-box, MAFbx,也称作Atrogin-1)和MurF-1(肌环指蛋白-1),在肌萎缩时显著激活。近年来,细胞凋亡在骨骼肌萎缩中的重要作用已经受到重视。文献报道,骼肌萎缩与肌细胞核的数量减少有关,而肌细胞核的数量主要是通过凋亡途径丢失的,骨骼肌萎缩与肌纤维凋亡有关。骨骼肌萎缩状态下,肌纤维促凋亡蛋白Bax、caspases的表达增加与抗凋亡蛋白Bcl-2的表达下调,说明线粒体途经的凋亡在分解代谢状态下发挥重要作用,并且其作用发生在蛋白降解之前。最新研究报道,Wnt7a还可以通过其受体Fzd7,激活蛋白激酶B/哺乳动物雷帕霉素靶蛋白(AKT/mTOR)信号通路,促进肌管肥大和肌纤维增粗。在给予杜氏进行性肌营养不良症(Duchenne muscular dystrophy,DMD)模型小鼠注射Wnt7a蛋白后,其肌纤维明显增粗,证实Wnt7a能促进肌肉组织生长和修复。虽然国内外大量研究文献都表明UPS活化、肌细胞凋亡增加及细胞内Wnt7a/Akt信号通路受损是导致骨骼肌萎缩的主要原因,但它们在CRF骨骼肌中的表达及作用尚缺乏研究,有必要探讨其在CRF骨骼肌萎缩中的作用及机制。CRF营养不良属于中医“关格”、“虚劳”等范畴。CRF营养不良多见面色萎黄,神疲乏力,纳差,消瘦,舌质淡,苔厚腻诸症。从祖国医学角度则认为与脾胃功能虚弱密切相关。盖脾为后天之本,气血生化之源,饮食入胃后,通过脾之运化,将精微物质化生气血,使之充养脏腑经络、四肢百骸、筋骨皮脉,即“中焦受气取汁变化而赤,是谓血”,亦谓“生化之源,则在于脾胃”。CRF时脾胃功能虚弱,水谷精微不化气血而成水湿浊毒。据此我们认为CRF营养不良其病在肾,关键在脾,脾胃功能失调贯彻CRF营养不良全过程。脾胃运化功能恢复,则后天补先天,有利于肾元恢复,气血生化有源,则营养状况得以改善。治疗中,鉴于CRF病机为本虚标实,虚实夹杂,当攻补兼施,正邪兼顾,治以补气血,益脾肾,泄湿浊,使补泄熔于一炉,扶正不留邪,祛邪不伤正。在此法指导下我们选用“人参养荣汤”加泻浊之品治疗CRF营养不良的患者,结果该方可以增加病人的食欲,提高血浆白蛋白,改善营养,提高患者的生活质量。肾衰营养胶囊是在“人参养荣汤”的基础上加减化裁并经科学的生产工艺研制而成,具有健脾补肾,益气养血,通腑泻浊之功效,在临床CRF营养不良的预防和治疗中取得客观疗效。本课题通过观察肾衰营养胶囊对CRF营养不良模型大鼠肾功能、肾脏病理改变、低蛋白血症、贫血以及骨骼肌萎缩的影响,检测大鼠骨骼肌UPS、细胞凋亡及Wnt7a/Akt信号通路的表达,初步探讨肾衰营养胶囊改善CRF营养不良及骨骼肌萎缩的机制,为肾衰营养胶囊防治CRF营养不良及骨骼肌萎缩,提供客观的理论实验依据。
二、方法：
取雄性SD大鼠,以5/6肾切除的方法制作CRF模型,将CRF的大鼠随机分为CRF营养不良模型组(Model)、肾衰营养胶囊组(SSYYJN)、和开同组(α-酮酸,KA)三组各10只。三组大鼠分别给予生理盐水、肾衰营养胶囊8g·kg-1·d-1、开同灌胃1g·kg-1·d-1,同时三组均继续给予低蛋白(4%酪蛋白)饲料喂养。另设假手术组(Control)10只,以生理盐水灌胃,正常蛋白饲料喂养。干预16周后,取大鼠血、尿进行血清肌酐(苦味酸法)、血清尿素氮(尿素酶法)、血清白蛋白(溴甲酚绿法)、Hb(碱化Hb比色法)、红细胞计数(血球计数仪)、血清转铁蛋白受体(免疫散射比浊法)、血清促红细胞生成素水平(多功能酶标仪)和24h尿蛋白(磺柳酸-硫酸钠比浊法)检测；留取肾脏组织包埋切片,进行Masson染色和电镜检测,观察肾脏纤维化的变化；获取胫骨前肌包埋切片,行HE染色测定肌纤维横截面积的变化；通过离体培养肌肉,检测单位时间内掺入的14+C-苯丙氨酸的放射性含量,分析蛋白合成代谢的变化；通过骨骼肌进行离体有氧培养,检测培养液中酪氨酸释放率,分析蛋白分解代谢的变化；免疫组化检测骨骼肌组织Ub蛋白的表达；免疫荧光检测骨骼肌组织MAFbx、MuRF-1蛋白；采用TUNEL法检测骨骼肌细胞凋亡的变化；Real-time PCR (RT-PCR)检测骨骼肌组织MAFbx、MuRF-1、FoxO1mRNA的表达；蛋白质印迹法(Western Blot)检测骨骼肌组织Wnt7a、phospho-Akt、phospho-mTOR、phospho-p70S6K、Bax、Bcl-2、 Cleaved caspase-3和GAPDH蛋白的表达；实验数据采用SPSS13.0统计软件进行分析。
三、结果：
1. SSYYJN对大鼠体重、胫骨前肌(Tibialis anterior, TA)湿重、干重及干重体重比的影响
术前各组大鼠体重无统计学差异(P>0.05)。体重在Model组显著下降(P<0.01),予以SSYYJN和KA干预可使其体重增加(P<0.01),且SSYYJN组较KA组增加更为显著(P<0.05)；TA湿重、干重在Model组显著下降(P<0.01),予以SSYYJN和KA干预可使其质量增加(P<0.01),但SSYYJN组和KA组间无显著性差异((P>0.05)；TA干重体重比在Model组显著减少(P<0.01),予以SSYYJN和KA干预可使其比值增加(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)；TA横截面积在Model组显著减少(P<0.01),予以SSYYJN和KA干预可使其大小增加(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)。
2. SSYYJN对大鼠血白蛋白、贫血相关指标的影响
血清白蛋白在Mode1组显著下降(P<0.01),予以SSYYJN和KA干预可使其水平增加(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)；血红蛋白(Hb)、红细胞(RBC)在Model组显著下降(P<0.01),予以SSYYJN和KA干预可使其水平增加(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)；血清转铁蛋白受体在Model组显著下降(P<0.01),予以SSYYJN和KA干预可使其水平增加(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)；血清促红细胞生成素在Model组显著下降(P<0.01),予以SSYYJN和KA干预可使其水平增加(P<0.01),且SSYYJN组较KA组增加更为显著(P<0.05)。
3. SSYYJN对大鼠肾功能指标的影响
术前各组血尿素氮、血肌酐无统计学差异。血尿素氮在Model组、SSYYJN和KA组显著下降(P<0.01),且KA组下降更为显著(P<0.01)；血肌酐在Model组显著升高(P<0.01),予以SSYYJN和KA干预可使其水平下降(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)。尿蛋白排泄率在Model组显著升高(P<0.01),且随时间进展而逐渐增加,予以SSYYJN和KA干预可使其水平下降(P<0.01)。
4. SSYYJN对大鼠肾脏病理的影响
Masson染色显示,Control组肾脏未见明显组织学异常,Model组则病变显著,出现明显肾小球硬化,小管间质病变更为显著,表现为小管扩张、萎缩、基底膜增厚,严重间质纤维化和炎症细胞浸润。给予SSYYJNIKA干预使GSI和TIS下降显著(P均
5. SSYYJN对大鼠骨骼肌蛋白合成和降解代谢的影响
通过对大鼠骨骼肌组织的4+C-苯丙氨酸释放率和酪氨酸掺入率检测发现,酪氨酸掺入率在Model组骨骼肌显著下降(P<0.01),提示Model组的蛋白合成代谢下降,予以SSYYJN和KA干预可使其掺入率增加(P<0.01),且KA组较SSYYJN组下降更为显著(P<0.01)；14+C-苯丙氨酸释放率在Model组显著增加(P<0.01),提示Model组的蛋白分解代谢加剧,予以SSYYJN和KA干预可使其释放率下降(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)。
6. SSYYJN对大鼠骨骼肌UPS、FoxO1mRNA表达的影响
首先通过对大鼠骨骼肌组织的免疫组化检测发现,Ub蛋白的AOD值在Model组显著增加(P<0.01),予以SSYYJN和KA干预可使其表达下降(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)；然后通过对大鼠骨骼肌组织的免疫荧光和Real-time PCR检测发现,MAFbx的平均荧光强度(MFI)、mRNA在Model组显著增加(P<0.01),予以SSYYJN和KA干预可使其表达下降(P<0.01),且KA组较SSYYJN组下降更为显著(P<0.01); MuRF-1MFI、mRNA在Model组显著增加(P<0.01),予以SSYYJN和KA干预可使其表达下降(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05); FoxO1mRNA在Model组显著增加(P<0.01),予以SSYYJN和KA干预可使其表达下降(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)。
7. SSYYJN对大鼠骨骼肌凋亡信号通路的影响
首先通过对大鼠骨骼肌组织的TUNEL检测发现,骨骼肌细胞凋亡率在Model组显著增加(P<0.01),予以SSYYJN和KA干预可使其凋亡率下降(P<0.01),且KA组较SSYYJN组下降更为显著(P<0.05)。然后通过对大鼠骨骼肌组织的Western Blot检测发现,Cleaved caspase-3蛋白在Model组显著增加(P<0.01),予以SSYYJN和KA干预可使其表达下降(P<0.01),且KA组较SSYYJN组下降更为显著(P<0.05); Bax蛋白在Model组显著增加(P<0.01),予以SSYYJN和KA干预可使其表达下降(P<0.01),且KA组较SSYYJN组下降更为显著(P<0.05)；Bcl-2蛋白在Model组显著减少(P<0.01),予以SSYYJN和KA干预可使其表达增加(P<0.01),且SSYYJN组较KA组增加更为显著(P<0.05)。
8. SSYYJN对大鼠骨骼肌Wnt7a-Akt/mTOR信号通路的影响
Western Blot检测发现,Wnt7a蛋白在Model组显著减少(P<0.01),予以SSYYJN和KA干预可使其表达增加(P<0.01),且SSYYJN组较KA组增加更为显著(P<0.05); Frizzled-7蛋白在Model组显著减少(P<0.01),予以SSYYJN和KA干预可使其表达增加(P<0.01或P<0.05),但SSYYJN组与KA组间差异无统计学意义(P>0.05); p-Akt蛋白在Model组显著减少(P<0.01),予以SSYYJN和KA干预可使其表达增加(P<0.01),且KA组较SSYYJN组增加更为显著(P<0.05)；p-mTOR蛋白在Model组显著下降(P<0.01),予以SSYYJN和KA干预可使其表达增加(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05); p-p70S6K蛋白在Model组显著下降(P<0.01),予以SSYYJN和KA干预可使其表达增加(P<0.01),但SSYYJN组与KA组间差异无统计学意义(P>0.05)。
四、结论：
1.CRF营养不良模型大鼠存在低白蛋白血症、贫血、骨骼肌萎缩等营养不良问题。SSYYJN可增加血清白蛋白、血红蛋白、红细胞计数、血清转铁蛋白受体、血清促红细胞生成素水平,改善骨骼肌萎缩。
2.CRF骨骼肌萎缩与UPS激活有关,该通路的激活可导致骨骼肌分解代谢旺盛,肌萎缩关键基因(MAFbx and MuRF1)表达升高,可能是CRF肌肉萎缩的机制之一。
3.CRF骨骼肌萎缩与细胞凋亡有关,促凋亡相关基因Bax的表达增加及抑凋亡相关基因Bcl-2的表达减少在骨骼肌细胞凋亡的发生机制中起着重要作用。
4.CRF骨骼肌萎缩与Wnt7a-Akt信号通路的下调有关,该通路的下调使Akt/mTOR和Akt/FoxO1信号途径异常,导致骨骼肌合成代谢下降和分解代谢增加,可能是CRF肌肉萎缩的另一个机制。
5. SSYYJN可以改善CRF骨骼肌萎缩,它可能是通过以下几个途径实现的：(1)抑制CRF骨骼肌UPS激活；(2)抑制CRF骨骼肌细胞凋亡,其机制可能是通过抑制促凋亡基因Bax的表达和增加抑凋亡基因Bcl-2的表达有关；(3)上调CRF骨骼肌Wnt7a/Akt信号通路。
Objective:
Malnutrition is one of the most common complications in chronic renal failure (CRF) patients, which not only has significant decreased storage protein, low serum albumin levels and anemia, but also highlighted the performance of progressive skeletal muscle atrophy, loss of lean body mass, decreased body storage protein, associated with poor prognosis in CRF patients. The etiology and pathogenesis of malnutrition with CRF is extremely complex. Reduced food intake, uremic toxin-induced nausea, vomiting, acidosis, and loss of plasma proteins and amino in peritoneal dialysis and hemodialysis patients could lead to malnutrition. In recent years, a number of studies have focused on protein metabolism, which contributed to our better understanding increase in protein degradation and decrease in protein synthesis, which played an important role in the pathogenesis of malnutrition with CRF. Studies have found that the ubiquitin proteasome system (UPS) involved in the abnormal protein and energy metabolism, showing80%-90%protein in cells were degraded by the UPS. UPS play an important role in the degradation of muscle proteins, two muscle-specific E3ligases:Fboxl muscular atrophy (MAFbx, also known as Atrogin-1), and Muscle-ring finger protein-1(MurF-1), were significantly increaed when muscle atrophy. In recent years, the apoptosis in the role of skeletal muscle atrophy has been focused on. Studies have found that the apoptosis decreased number of nucleus, which were related with muscle atrophy. The increased in pro-apoptotic Bax, caspases protein and the decreased in Bcl-2protein induced apoptosis in a catabolic state. It reported that Wnt7a binding to Fzd7directly activated the Akt/mTOR growth pathway, thereby inducing myofibre hypertrophy in differentiated myofibres. Duchenne muscular dystrophy (DMD) in mice after injecting Wnt7a protein, significantly enlarged its muscle fibers, which confirmed Wnt7a can promote muscle atrophy. Although studies suggested that activation of UPS, increase in muscle cell apoptosis and downregulation of Wnt7a-Akt/mTOR signaling pathways were the main reason which leading to muscle atrophy, but the expression of them in skeletal muscle of CRF has been unknown, it is necessary to explore its role in the muscle atrophy with CRF. Capsule of Shengshuai Yingyang (SSYYJN) was empirical prescription based on "Renshen Yangrong tang", malnutrition with CRF patients were treated with SSYYJN for many years. Our pre-clinical observation showed that SSYYJN has a better clinical curative effect on malnutrition of CRF patients. To profoundly study the role of UPS, apoptosis and Wnt7a-AKT/mTOR signaling pathwany in the mechanism of skeletal muscle atrophy, and to explore the role of Wnt7a in the pathogenesis of in CRF with malnutrition and the molecular mechanism of SSYYJN for treatment of CRF with malnutrition.
Methods:
Male rats were randomly assigned to either the5/6nephrectomy group or the sham-operated group. Those5/6nephrectomy rats were randomized into CRF model group, KA group, and SSYYJN group. The three group were fed with4%casein. The sham operated group as control group. The KA group and SSYYJN group were treated with Ketosteril and capsule of Shenshuai Yingyang for16weeks. The general status of rats, body weight was observed during the experiment. During and after treatment, the blood parameters such as ALB, BUN, Scr,24hUpro, Hb, PA, Tf, and EPO were detected, and the pathological change of renal tissue were stained by Masson and electron microscopy. The size of tibia anterior (TA) muscle was measured under HE staining and calculated with software. Protein synthesis was measured in vitro in the soleus muscle using the incorporation of14+C-phenylalanine. Protein degradation was measured using tyrosine release from the isolated muscle. The expression of Ub protein was measured by immunohistochemistry. The expression of MAFbx, MuRF-1was measured by immunofluorescence. DNA strand breaks were assessed by fluorescent labelling of terminal dUTP nick end labelling (TUNEL) using the onestep TUNEL apoptosis assay kit, TUNEL-positive cells were counted, and then their densities were calculated. The mRNA expression of MAFbx, MuRF-1and FoxO1were measured by RT-PCR. Expression of Wnt7a、phospho-Akt、 phospho-mTOR、phospho-p70S6K、Bax、Bc1-2、Cleaved caspase-3and GAPDH were detected by Western Blot.
Results:
1. The effects of SS YYJN treatment on body weight and TA muscle weight
The body weight of each group has no significant differences before the intervention. After the intervention, the body weight of Model group was significantly heavier than control group. The body weights of SSYYJN and KA groups were substantially higher than the Model Group (P<0.01), and the body weight of SSYYJN was higher than the KA Group (P<0.05). The wet and dry weights of TA muscle from Model group was a significant reduction (P<0.01) when compared with the Control Group. SSYYJN group and KA groups were significantly higher ((P<0.01) than the Model group, but no statistical differences were observed between the between SSYYJN and KA groups (P>0.05). The dry weight of TA muscle corrected for body weight from Model group was a significant reduction (P<0.01) when compared with the Control Group. SSYYJN group and KA groups were significantly higher ((P<0.01) than the Model group, but no statistical differences were observed between the between SSYYJN and KA groups (P>0.05). The mean cross-sectional area of TA muscle from Model group was a significant reduction (P<0.01) when compared with the Control Group. SSYYJN group and KA groups were significantly higher ((P<0.01) than the Model group, but no statistical differences were observed between the between SSYYJN and KA groups (P>0.05).
2. The effects of SSYYJN treatment on serum albumin and Anemia-related indicators.
Results showed that the level of serum albumin from Model Group was significantly lower than the Control group (P<0.01), the level of serum albumin from SSYYJN and KA groups were higher than the Model group, but no statistical differences were observed between the between SSYYJN and KA groups. Model Group Hb and RBC count is significantly lower than control group (P<0.01), SSYYJN and KA Hb and RBC count higher than the Model group, SSYYJN group Hb and RBC count higher than the KA Group (P<0.01). Serum transferrin receptor was lower in the Model groups than in the Control group (P<0.01), the level of serum transferrin receptor from SSYYJN and KA groups were higher than the Model group, but no statistical differences were observed between the between SSYYJN and KA groups. Serum Erythropoietin was lower in the Model groups than in the Control group (P<0.01), the level of Erythropoietin from SSYYJN groups was higher than the Model and KA groups (P<0.01).
3. The effects of SSYYJN treatment on proteinuria and renal function
Blood biochemical values of each group before the intervention has no significant difference. Intervention the values of blood urea nitrogen in the Model group was significantly lower than Control group (P<0.01), the values of blood urea nitrogen from SSYYJN and KA groups were lower than the Model group. The level of serum creatinine from Model Group was significantly higher than the Control group (P<0.01), the level of serum albumin from SSYYJN and KA groups were higher than the Model group, but no statistical differences were observed between the between SSYYJN and KA groups. The levels of urinary albumin excretion was highest in the Model group, and increased progressively with time, show progressive deterioration of renal function, and SSYYJN reduced the urinary protein.
4. The effects of SSYYJN treatment on renal tissue damage
Masson stain showed that no histologic abnormalities of the kidneys were observed in the control group. In the5/6nephrectomy groups, glomerulosclerosis and interstitial fibrosis progressed, especially in the Model group (P<0.01). Compared with the Model group, the SSYYJN and KA group showed more decrease in the glomerulosclerosis index and interstitial fibrosis score (P<0.01), but no statistical differences were observed between the between SSYYJN and KA groups (P>0.05).
5. The effects of SSYYJN treatment on muscle protein synthesis and protein degradation.
The rate of protein synthesis was significantly lower in the Model group than that in the Control group (PO.01), and SSYYJN and KA supplementation increased the rate of protein synthesis when compared with the Model group (P<0.01). The rates of protein degradation were significantly higher in the Model groups than in the Control group (P<0.01), and the rates of protein degradation from SSYYJN and KA groups were lower than the Model group, but no statistical differences were observed between the between SSYYJN and KA (P>0.05).
6. The effects of SSYYJN treatment on UPS
The protein content of ubiquitin was examined by immunohistochemical analysis. Marked immune positive staining of ubiquitin was found to be localised in the cytoplasmic region of scattered muscle fibres in the muscle of the5/6nephrectomy groups, especially in the Model group (P<0.01). The percentage of ubiquitin-positive areas calculated through semi-quantitative analysis was found to be significantly larger in the SSYYJN and KA groups than in the Model groups (P<0.01), but no statistical differences were observed between the between SSYYJN and KA (P>0.05). The expression of MAFbx and MuRF1conducive to skeletal muscle atrophy was examined by immunofluorescent staining. The mean fluorescence intensity of MAFbx and MuRF1remarkably increased in the Model group than that in the Control group (P<0.01), while the intensity was tamed with SSYYJN and KA treatment when compared with the Model group and SSYYJN group was higher than KA group. The mRNA expression of MAFbx and MuRF1assessed by RT-PCR showed significant up-regulation in the Model group but down-regulation with KA and SSYYJN intervention and SSYYJN group was higher than KA group, which is consistent with the results of immunofluorescence studies of MAFbx and MuRF1. The mRNA expression of FoxO1assessed by RT-PCR showed significant up-regulation in the Model group but down-regulation with KA and SSYYJN intervention but no statistical differences were observed between the between SSYYJN and KA groups.
7. The effects of SSYYJN treatment on apoptosis signaling pathway
DNA fragments and the level of cleaved caspase-3in skeletal muscle were evaluated by TUNEL staining and immunoblotting. TUNEL-positive nuclei of TA muscle was observed with significant increases in the Model group as compared with the Control groups (p<0.01), but distinct decrease in the SSYYJN and KA group as compared with the Model group, distinct decrease in the KA group as compared with the SSYYJN group. The semi-quantification of cleaved caspase-3also showed a similar result with TUNEL staining. Furthermore, to determine whether Bcl-2family proteins would regulate caspase-3dependent apoptotic pathway, additional immunoblotting was performed to examine the expression of Bax and Bcl-2protein. Findings demonstrated upregulated level of Bax but downregulated level of Bcl-2in the Model group as compared with the Control group (p<0.01), while upregulated level of Bcl-2but downregulated level of Bax with SSYYJN and KA treatment as compared with the Model group (p<0.01).
8. The effects of SSYYJN treatment on the Wnt7a/Akt/p70S6K signaling pathway
Evidence has been provided indicating that Wnt7a directly activated the Akt/p70S6K pathway to induce myofibre hypertrophy. We examined Wnt7a, p-Akt, p-mTOR and p-p70S6K protein levels by immunoblotting. The data indicate a lower level of Wnt7a protein in the Model group than that in the Control group (p<0.01), whereas treatment with SSYYJN and KA increased the level of Wnt7a protein (p<0.01), SSYYJN increased the level of Wnt7a protein were significantly higher than KA. Meanwhile, downward trend in Akt, mTOR and p70S6K phosphorylation in the Model group was also observed compared with the Control groups (P<0.01), but SSYYJN and KA supplementation prompted an increase as compared with the Model group (P<0.01). SSYYJN increased the level of p-Akt protein than KA, no statistical differences in the protein level of p-mTOR and p-p70S6K were between the SSYYJN and KA groups.
Conclusion:
1. SSYYJN can improve malnutrition in CRF by increasing the level of blood serum albumin, hemoglobin, red blood cell count, transferrin receptor and serum erythropoietin.
2. The increases in MAFbx and MuRF1expression are linked to the proposed role of the UPS associated with muscle atrophy in CRF.
3. Apoptosis is closely related to muscle atrophy in CRF. The increase of Bax gene expression and decrease of Bcl-2gene expression induced apoptosis in skeletal muscle.
4. Down-regulation of the Wnt7a/Akt signaling pathway is closely related to muscle atrophy in CRF, which leading to abnormal of Akt/mTOR and Akt/FoxO1signaling pathway were correlated with lower rates of protein synthesis and higher rates of protein degradation.
5. SSYYJN can effectively improve muscle atrophy in CRF. The mechanism may be as follows:(1) SSYYJN can prevent the activation of the UPS in CRF skeletal muscle;(2) SSYYJN can inhibit muscular cell apoptosis in CRF skeletal muscle;(3) SSYYJN can reverse declining expression of Wnt7a/Akt signaling pathway in CRF skeletal muscle.
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