侯洪濤,裘艷梅,張建,胡義亭,王玉珍
(1 河北省人民醫(yī)院,石家莊050051;2 河北醫(yī)科大學(xué)第二醫(yī)院)
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利拉魯肽對(duì)非酒精性脂肪肝大鼠IR及SOCS-3表達(dá)的影響
侯洪濤1,裘艷梅2,張建1,胡義亭1,王玉珍1
(1 河北省人民醫(yī)院,石家莊050051;2 河北醫(yī)科大學(xué)第二醫(yī)院)
目的 探討利拉魯肽對(duì)非酒精性脂肪肝(NAFLD)的治療作用及其機(jī)制。方法 制備NAFLD大鼠模型20只,隨機(jī)分為高脂組、利拉魯肽組各10只,另取10只正常大鼠作為對(duì)照組。高脂組、利拉魯肽組高脂飼料飼養(yǎng)16周,對(duì)照組普通飼料飼養(yǎng)16周。利拉魯肽組飼養(yǎng)12周末腹腔注射利拉魯肽 600 μg/(kg·d)4周;飼養(yǎng)第16周末,三組均心臟取血,檢測(cè)血清ALT、AST、TG、TC和空腹血糖(FPG)、空腹胰島素(FINS)水平,計(jì)算胰島素抵抗指數(shù)(HOMA-IR)。處死大鼠,稱量體質(zhì)量及肝質(zhì)量,計(jì)算肝指數(shù);取部分肝臟,HE染色,光鏡下觀察肝組織病理變化;另取部分肝組織,RT-PCR法檢測(cè)細(xì)胞因子信號(hào)轉(zhuǎn)導(dǎo)抑制物3(SOCS-3)mRNA表達(dá),分析SOCS-3 mRNA表達(dá)與HOMA-IR的關(guān)系。結(jié)果 與對(duì)照組比較,高脂組肝脂數(shù)、ALT、AST、TG、TC、FPG、FINS及HOMA-IR均明顯升高(P均<0.05);與高脂組比較,利拉魯肽組上述指標(biāo)均明顯降低(P均<0.05)。對(duì)照組肝組織無異常病理改變;高脂組肝細(xì)胞排列紊亂,細(xì)胞內(nèi)有較多脂滴,部分肝細(xì)胞變性,有炎性細(xì)胞浸潤(rùn);利拉魯肽組肝細(xì)胞脂肪變明顯輕于高脂組。高脂組肝組織SOCS-3 mRNA相對(duì)表達(dá)量明顯高于對(duì)照組、利拉魯肽組(P均<0.05),對(duì)照組、利拉魯肽組比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。直線相關(guān)分析顯示,肝組織SOCS-3 mRNA表達(dá)與HOMA-IR呈正相關(guān)(r=0.445,P<0.05)。結(jié)論 利拉魯肽治療NAFLD有一定效果,其作用機(jī)制可能與減輕胰島素抵抗及抑制SOCS-3 mRNA表達(dá)有關(guān)。
非酒精性脂肪肝;胰島素抵抗;胰高血糖素樣肽1;細(xì)胞因子信號(hào)轉(zhuǎn)導(dǎo)抑制物3
非酒精性脂肪肝(NAFLD)是與代謝性疾病相關(guān)的肝臟疾病,與胰島素抵抗(IR)、糖尿病、肥胖、高脂血癥等因素密切相關(guān),被認(rèn)為是代謝綜合征的肝臟表現(xiàn)。近年來我國(guó)NAFLD發(fā)病率顯著升高,并呈年輕化趨勢(shì)。NAFLD的發(fā)病機(jī)制尚未完全闡明,二次打擊學(xué)說是NAFLD發(fā)病機(jī)制的經(jīng)典學(xué)說[1]。IR被認(rèn)為是導(dǎo)致肝臟脂質(zhì)過度沉積的原發(fā)病因,在NAFLD的發(fā)生、發(fā)展過程中具有重要作用[2]。近年研究發(fā)現(xiàn),細(xì)胞因子信號(hào)轉(zhuǎn)導(dǎo)抑制物3(SOCS-3)可影響胰島素信號(hào)轉(zhuǎn)導(dǎo),導(dǎo)致脂肪酸代謝紊亂,并參與IR及NAFLD的發(fā)病過程[3]。胰高血糖素樣肽1(GLP-1)能改善IR及肝臟脂質(zhì)沉積,已被臨床用于NAFLD的治療[4]。利拉魯肽是GLP-1類似物,是一種治療糖尿病的新型藥物,其能否用于治療NAFLD尚不十分清楚。2015年7~11月,本研究探討了利拉魯肽對(duì)NAFLD的治療作用及其機(jī)制。
1.1 材料 健康雄性SD大鼠32只,6周齡,體質(zhì)量140~150 g,購(gòu)自河北醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心,動(dòng)物許可證號(hào):SCXK(冀)2013-1-003。飼養(yǎng)環(huán)境:溫度22~24 ℃,濕度50%左右,12 h晝夜交替,分籠飼養(yǎng)。普通飼料及豬油,由河北醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心提供;膽固醇,購(gòu)自河北醫(yī)科大學(xué)海森醫(yī)藥有限公司;利拉魯肽,購(gòu)自丹麥諾和諾德公司;胰島素酶聯(lián)免疫試劑盒,購(gòu)自南京建成生物工程研究所;TRIzol試劑盒、逆轉(zhuǎn)錄體系、擴(kuò)增體系及引物,購(gòu)自北京賽百盛基因技術(shù)有限公司。
1.2 模型制備及干預(yù) 所有大鼠適應(yīng)性飼養(yǎng)1周,隨機(jī)取22只給予高脂飼料(普通飼料88%、豬油10%、膽固醇2%)飼養(yǎng)制備NAFLD模型,飼養(yǎng)12周隨機(jī)處死2只,取肝組織制作切片,行HE染色,觀察到肝細(xì)胞出現(xiàn)明顯脂肪變性,證實(shí)NAFLD模型制備成功。將20只成模大鼠隨機(jī)分為高脂組、利拉魯肽組,每組10只;兩組繼續(xù)高脂飼料飼養(yǎng),利拉魯肽組腹腔注射利拉魯肽600 μg/(kg·d),高脂組腹腔注射等體積生理鹽水,連續(xù)4周。剩余10只大鼠作為對(duì)照組,予普通飼料飼養(yǎng)。三組均飼養(yǎng)至第16周。
1.3 相關(guān)指標(biāo)觀察
1.3.1 血生化學(xué)指標(biāo) 飼養(yǎng)至第16周末,腹腔注射麻醉,迅速心臟取血,靜置2 h,以3 000 r/min離心10 min,取上層血清,-70 ℃冰箱凍存?zhèn)溆?。采用全自?dòng)生化分析儀檢測(cè)血清TG、TC、ALT、AST,快速血糖儀測(cè)定空腹血糖(FPG);酶聯(lián)免疫法測(cè)定空腹胰島素(FINS),計(jì)算胰島素抵抗指數(shù)(HOMA-IR)。HOMA-IR=FPG×FINS/22.5。
1.3.2 肝組織病理檢查 飼養(yǎng)至第16周末,處死大鼠,迅速取出肝臟,觀察肝臟大體情況,并稱質(zhì)量,計(jì)算肝指數(shù)(肝指數(shù)=肝臟濕質(zhì)量/體質(zhì)量×100%)。每只大鼠取相同部位肝組織,4%多聚甲醛固定,常規(guī)脫水,石蠟包埋,4 μm厚切片,HE染色,光鏡下觀察肝組織病理變化。
1.3.3 肝組織SOCS-3 mRNA表達(dá) 取凍存肝組織100 mg,置于玻璃勻漿器中,加入TRIzol 1 mL冰上研磨,徹底勻漿。按照TRIzol試劑盒說明書提取肝組織總RNA,在逆轉(zhuǎn)錄酶的催化下合成cDNA,以cDNA為模板在Taq DNA聚合酶催化下進(jìn)行PCR擴(kuò)增。擴(kuò)增產(chǎn)物150 bp。擴(kuò)增條件:95 ℃預(yù)變性3 min,95 ℃變性30 s,55 ℃退火40 s,72 ℃延伸60 s,循環(huán)40次,最后72 ℃延伸10 min。PCR產(chǎn)物在2%的瓊脂糖凝膠中電泳,凝膠成像儀采集圖像,PCR儀獲得產(chǎn)物Ct值,采用2-ΔΔCt法計(jì)算SOCS-3 mRNA相對(duì)表達(dá)量。
2.1 各組一般狀態(tài)、體質(zhì)量及肝指數(shù)比較 三組均全部存活;對(duì)照組皮毛光澤,靈活好動(dòng);高脂組不喜活動(dòng),皮毛光澤度差;利拉魯肽組對(duì)外界反應(yīng)靈敏,皮毛光澤度明顯好于高脂組。各組體質(zhì)量、肝質(zhì)量及肝指數(shù)比較見表1。
2.2 各組血生化指標(biāo)比較 見表2。
2.3 各組肝組織病理觀察 光鏡下對(duì)照組肝小葉結(jié)構(gòu)清晰,肝細(xì)胞索呈放射狀排列, 胞質(zhì)均勻,無脂滴浸潤(rùn),匯管區(qū)及小葉內(nèi)無炎性細(xì)胞浸潤(rùn);高脂組肝細(xì)胞排列紊亂,細(xì)胞內(nèi)有較多脂滴,部分肝細(xì)胞變性,有炎性細(xì)胞浸潤(rùn);利拉魯肽組肝細(xì)胞脂肪變性明顯改善,肝細(xì)胞內(nèi)有少量脂肪滴,炎性細(xì)胞浸潤(rùn)改善。
表1 各組體質(zhì)量、肝質(zhì)量及肝指數(shù)比較±s)
注:與對(duì)照組比較,*P<0.05;與高脂組比較,#P<0.05。
表2 各組血生化指標(biāo)比較±s)
注:與對(duì)照組比較,*P<0.05;與高脂組比較,#P<0.05。
2.4 各組肝組織SOCS-3 mRNA表達(dá)比較 對(duì)照組肝組織SOCS-3 mRNA相對(duì)表達(dá)量為1.30±0.44,高脂組為3.06±1.62,利拉魯肽組為2.00±0.75。高脂組肝組織SOCS-3 mRNA相對(duì)表達(dá)量明顯高于對(duì)照組、利拉魯肽組(P均<0.05),而對(duì)照組、利拉魯肽組比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。
2.5 肝組織SOCS-3 mRNA表達(dá)與HOMA-IR的關(guān)系 直線相關(guān)分析顯示,各組肝組織SOCS-3 mRNA表達(dá)與HOMA-IR呈正相關(guān)(r=0.45,P<0.05)。
NAFLD是與代謝性疾病相關(guān)的肝臟疾病,其發(fā)生、發(fā)展受代謝、遺傳、環(huán)境、腸道微生物和生活方式等多種因素調(diào)節(jié),與IR、肥胖、高脂血癥等密切相關(guān)。NAFLD的發(fā)病機(jī)制目前尚未完全清楚,其理論研究還停留在假說階段,“二次打擊學(xué)說”是目前最為人們接受和認(rèn)可的觀點(diǎn)。第一次打擊是以IR為中心環(huán)節(jié)導(dǎo)致的肝細(xì)胞脂肪變性、肝臟脂質(zhì)沉積;在此基礎(chǔ)上發(fā)生的,以線粒體反應(yīng)氧體系為核心的氧化應(yīng)激和脂質(zhì)過氧化對(duì)肝臟的損傷是NAFLD發(fā)病中的第二次打擊。
IR是NAFLD發(fā)病的中心環(huán)節(jié),貫穿于NAFLD的全過程,不僅參與首次打擊,也參與二次打擊。IR引起NAFLD的可能機(jī)制:IR可減弱胰島素對(duì)脂肪代謝的調(diào)節(jié),使外周脂肪分解增強(qiáng),血清中游離脂肪酸(FFA)濃度上升,F(xiàn)FA隨血液循環(huán)進(jìn)入肝臟增多,導(dǎo)致肝內(nèi)TG大量合成并堆積;肝臟增多的FFA會(huì)導(dǎo)致肝細(xì)胞線粒體氧化超載,從而加重肝細(xì)胞內(nèi)脂肪變性[5]。此外,增多的FFA還可通過抑制胰島素信號(hào)轉(zhuǎn)導(dǎo),減少胰島素清除,而加重IR。
SOCSs是一類具有抑制Janus 激酶信號(hào)轉(zhuǎn)導(dǎo)子與轉(zhuǎn)錄激活子(JAK/STAT)信號(hào)轉(zhuǎn)導(dǎo)的蛋白質(zhì)家族,目前對(duì)SOCS-1和SOCS-3的研究較多。SOCS-3在體內(nèi)廣泛分布于肝、腎、腦、心等組織器官[6]。SOCS-3由225個(gè)氨基酸組成,分子量約為24.75 ku。人類SOCS-3基因位于17q25.3,長(zhǎng)度為850 bp,與大鼠、小鼠的同源性約為90%。正常組織中SOCS-3低表達(dá),許多細(xì)胞因子,如紅細(xì)胞生成素、生長(zhǎng)激素、IL-6等可誘導(dǎo)SOCS-3表達(dá)。這些細(xì)胞因子誘導(dǎo)SOCS-3表達(dá)后,其表達(dá)產(chǎn)物又抑制細(xì)胞因子介導(dǎo)的JAK/STAT信號(hào)通路。胰島素信號(hào)傳導(dǎo)受阻或減弱是IR的主要原因之一,SOCS-3通過多種途徑參與IR:①與STAT5b競(jìng)爭(zhēng)性結(jié)合胰島素受體,抑制STAT5b的磷酸化及其胰島素受體底物的磷酸化,使下游的胰島素信號(hào)轉(zhuǎn)導(dǎo)受阻[7];②通過SH2結(jié)構(gòu)域與信號(hào)蛋白受體上磷酸化的酪氨酸結(jié)合,干擾胰島素信號(hào)的轉(zhuǎn)導(dǎo)[8];③與信號(hào)蛋白耦聯(lián),從而被蛋白酶體降解,抑制胰島素信號(hào)的轉(zhuǎn)導(dǎo)[9]。SOCS-3還參與抵抗素誘導(dǎo)的IR,抵抗素可使SOCS-3表達(dá)升高,并促進(jìn)其與胰島素受體結(jié)合,從而抑制胰島素信號(hào)的傳導(dǎo)[10]。應(yīng)用拮抗SOCS-3的寡核苷酸對(duì)db/db小鼠進(jìn)行干預(yù),肝組織SOCS-3表達(dá)下降,脂肪肝程度明顯減輕[11]。本研究結(jié)果顯示,對(duì)照組肝組織SOCS-3 mRNA低表達(dá),高脂組SOCS-3 mRNA表達(dá)顯著高于對(duì)照組,說明SOCS-3參與了NAFLD的發(fā)病過程。
GLP-1是由腸道L細(xì)胞分泌的腸道激素,具有促進(jìn)胰島素釋放、延緩胃排空、抑制胰高血糖素的釋放等作用。GLP-1的生物學(xué)作用是靠GLP-1和細(xì)胞上的GLP-1受體結(jié)合來實(shí)現(xiàn),GLP-1受體在人體內(nèi)主要分布于胰島α、β細(xì)胞,胃、小腸、肺、心臟、腎臟、垂體及下丘腦等組織。研究表明,人肝細(xì)胞亦有GLP-1受體表達(dá)[12]。利拉魯肽和艾塞那肽均為GLP-1類似物,均通過增加胰島素分泌、改善胰島細(xì)胞功能、減少食物攝入量等多種機(jī)制發(fā)揮降糖作用,已廣泛應(yīng)用于2型糖尿病的治療[13]。近年研究發(fā)現(xiàn),GLP-1類似物對(duì)代謝綜合征有治療作用,NAFLD是代謝綜合征在肝臟的表現(xiàn)。有研究發(fā)現(xiàn),應(yīng)用艾塞那肽干預(yù)ob/ob小鼠,可使小鼠體質(zhì)量減輕,改善胰島素敏感性,減輕肝內(nèi)脂質(zhì)沉積[14~18]。本研究結(jié)果顯示,與對(duì)照組比較,高脂組體質(zhì)量、肝指數(shù)明顯升高,肝臟脂肪變性及炎癥程度明顯增加,其血清ALT、AST、FPG、FINS及HOMA-IR明顯升高,表明IR在NAFLD的發(fā)生、進(jìn)展過程中具有重要作用。而利拉魯肽組上述指標(biāo)及肝組織SOCS-3 mRNA表達(dá)均明顯低于高脂組,說明利拉魯肽能夠改善NAFLD大鼠肝臟脂肪變性。
綜上所述,利拉魯肽對(duì)NAFLD有治療作用,其作用機(jī)制可能與減輕IR及抑制SOCS-3 mRNA表達(dá)有關(guān)。
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Effects of liraglutide on insulin resistance and SOCS-3 expression in rats with nonalcoholic fatty liver disease
HOUHongtao1,QIUYanmei,ZHANGJian,HUYiting,WANGYuzhen
(1People′sHospitalofHebeiProvince,Shijiazhuang050051,China)
Objective To investigate the effects of liraglutide (Lira) on insulin resistance and expression of SOCS-3 in rats with nonalcoholic fatty liver disease (NAFLA). Methods Twenty NAFLD rat models were prepared and then randomly divided into high fat group (HF) and HF+Lira group. Meanwhile, another 10 normal rats were selected as the normal control (NC) group. HF group, HF+Lira group were given high fat diet for 16 weeks. After 12 weeks of high fat feeding in HF+Lira group, 600 μg/(kg·d) Lira was administered by intraperitoneal injection for 4 weeks. At the end of the 16 weeks, the rats were killed. The changes of liver pathology were observed by optical microscope. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total triglyceride (TG), cholesterol (TC) and fasting blood glucose (FPG) were detected by automatic biochemical analyzer. The fasting insulin (FINS) was determined by radioimmunoassay, and insulin resistance index (HOMA-IR) was assessed by homeostasis mode assessment. The liver expression of SOCS-3 mRNA was detected by RT-PCR. The relationship between SOCS-3 mRNA and HOMA-IR was analyzed. Results Compared with NC group, liver index, HOMA-IR, and the levels of ALT, AST, TG, TC, FPG and FINS were significantly increased in HF group (allP<0.05); compared with HF group, the above indexes of the HF+Lira group were all decreased, and the difference was statistically significant (allP<0.05). No abnormal pathological changes in the liver tissues of the NC group. In the HF group, the liver cells were arranged in disorder, there were more lipid droplets in the cells, and some hepatocytes degenerated and had inflammatory cells infiltration. The hepatic steatosis in the HF+Lira group was lighter than that in the HF group. The expression level of SOCS-3 mRNA in HF group was significantly higher than that in NC group and the HF+Lira group (allP<0.05); and no significant difference was found between the NC group and the HF+Lira group (P>0.05). HOMA-IR was positively correlated with of the expression of SOCS-3 mRNA (r=0.445,P<0.05). Conclusion Lira has certain effect in treatment of NAFLD, whose mechanism may be related to the decrease of IR and inhibition of SOCS-3 mRNA expression.
nonalcoholic fatty liver; insulin resistance; glucagon-like peptide 1; suppressor of cytokine signaling 3
河北省醫(yī)學(xué)科學(xué)研究重點(diǎn)課題計(jì)劃(ZL20150117)。
侯洪濤(1980-),男,碩士,主治醫(yī)師,研究方向?yàn)橄到y(tǒng)疾病診治。E-mail: llvzi@126.com
10.3969/j.issn.1002-266X.2016.40.005
R575.5
A
1002-266X(2016)40-0017-04
2016-01-18)