目的 探讨白藜芦醇对糖尿病大鼠肾皮质蛋白烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)、葡萄糖调节蛋白78( GRP78)表达的影响,以研究白藜芦醇对糖尿病大鼠肾皮质氧化应激损伤的保护作用.方法 将15只SD雄性大鼠应用链脲佐菌素(STZ)制造糖尿病大鼠模型.13周后将造模成功的12只大鼠随机分为糖尿病组(DM组,n=6)和白藜芦醇干预组(DR,n=6),以6只健康SD大鼠作为对照组(NC组).DR组大鼠给予白藜芦醇10 mg·kg-1·d-1每日固定时间连续灌胃2周,DM组用等体积0.5％羧甲基纤维素钠溶液灌胃,至第14周结束.第14周结束时,检测3组大鼠的血糖、体重、血肌酐、血尿素氮、24 h尿微量白蛋白.取肾组织,制石蜡切片做PAS染色观察肾小球肾小管形态学变化.取10％肾皮质组织匀浆后测定丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)水平.Western blotting法检测肾皮质NOX4和GRP78蛋白表达的改变.结果 与NC组相比,DM组大鼠血糖、血肌酐、血尿素氮、24 h尿微量白蛋白均显著升高(t=- 52.324、- 20.487、- 20.724、- 55.476,均P＜0.0167),而体重则明显下降(t=5.820,P ＜0.0167)；白藜芦醇干预后,DR组大鼠24 h尿微量白蛋白、血肌酐、血尿素氮均比DM组呈好转趋势(t=13.963、7.849、8.678,均P＜0.0167)；而DR组体重和血糖与DM组相比差异无统计学意义(t=-1.767、1.876,均P＞0.0167).与NC组相比,DM组MDA水平明显增加(t=- 10.661,P＜0.0167),而SOD、CAT水平降低(t=8.124、8.222,均P＜0.0167)；而与DM组相比,DR组SOD水平升高(t=- 12.309,p＜0.0167),MDA水平降低(t=4.475,P＜0.0167),2组CAT活力差异无统计学意义(t=-3.029,P＞0.0167).肾脏组织PAS染色显示NC组大鼠基底膜完整无增厚,DM组大鼠肾小球系膜基质增多,系膜细胞增生,DR组则较DM组减轻.Western blotting显示DM组血管组织中NOX4及GRP78蛋白表达上调(0.49±0.05、1.47±0.04),均高于对照组NC组(0.09±0.001、0.84±0.012,t=- 14.255、-25.179,均P＜ 0.0167)；DR组NOX4蛋白表达(0.30 ±0.073)仍高于NC组(t=-5.125,P＜0.0167)；GRP78蛋白(0.60 ±0.034)则低于NC组(t=- 28.017,P＜0.0167).结论 糖尿病大鼠肾皮质上存在着明显的氧化应激损伤,白藜芦醇可能通过抑制GRP78蛋白的表达从而影响内质网应激,抑制NOX4蛋白的表达减轻氧化应激对糖尿病大鼠早期肾脏损害.
目的 探討白藜蘆醇對糖尿病大鼠腎皮質蛋白煙酰胺腺嘌呤二覈苷痠燐痠氧化酶4(NOX4)、葡萄糖調節蛋白78( GRP78)錶達的影響,以研究白藜蘆醇對糖尿病大鼠腎皮質氧化應激損傷的保護作用.方法 將15隻SD雄性大鼠應用鏈脲佐菌素(STZ)製造糖尿病大鼠模型.13週後將造模成功的12隻大鼠隨機分為糖尿病組(DM組,n=6)和白藜蘆醇榦預組(DR,n=6),以6隻健康SD大鼠作為對照組(NC組).DR組大鼠給予白藜蘆醇10 mg·kg-1·d-1每日固定時間連續灌胃2週,DM組用等體積0.5％羧甲基纖維素鈉溶液灌胃,至第14週結束.第14週結束時,檢測3組大鼠的血糖、體重、血肌酐、血尿素氮、24 h尿微量白蛋白.取腎組織,製石蠟切片做PAS染色觀察腎小毬腎小管形態學變化.取10％腎皮質組織勻漿後測定丙二醛(MDA)、超氧化物歧化酶(SOD)、過氧化氫酶(CAT)水平.Western blotting法檢測腎皮質NOX4和GRP78蛋白錶達的改變.結果 與NC組相比,DM組大鼠血糖、血肌酐、血尿素氮、24 h尿微量白蛋白均顯著升高(t=- 52.324、- 20.487、- 20.724、- 55.476,均P＜0.0167),而體重則明顯下降(t=5.820,P ＜0.0167)；白藜蘆醇榦預後,DR組大鼠24 h尿微量白蛋白、血肌酐、血尿素氮均比DM組呈好轉趨勢(t=13.963、7.849、8.678,均P＜0.0167)；而DR組體重和血糖與DM組相比差異無統計學意義(t=-1.767、1.876,均P＞0.0167).與NC組相比,DM組MDA水平明顯增加(t=- 10.661,P＜0.0167),而SOD、CAT水平降低(t=8.124、8.222,均P＜0.0167)；而與DM組相比,DR組SOD水平升高(t=- 12.309,p＜0.0167),MDA水平降低(t=4.475,P＜0.0167),2組CAT活力差異無統計學意義(t=-3.029,P＞0.0167).腎髒組織PAS染色顯示NC組大鼠基底膜完整無增厚,DM組大鼠腎小毬繫膜基質增多,繫膜細胞增生,DR組則較DM組減輕.Western blotting顯示DM組血管組織中NOX4及GRP78蛋白錶達上調(0.49±0.05、1.47±0.04),均高于對照組NC組(0.09±0.001、0.84±0.012,t=- 14.255、-25.179,均P＜ 0.0167)；DR組NOX4蛋白錶達(0.30 ±0.073)仍高于NC組(t=-5.125,P＜0.0167)；GRP78蛋白(0.60 ±0.034)則低于NC組(t=- 28.017,P＜0.0167).結論 糖尿病大鼠腎皮質上存在著明顯的氧化應激損傷,白藜蘆醇可能通過抑製GRP78蛋白的錶達從而影響內質網應激,抑製NOX4蛋白的錶達減輕氧化應激對糖尿病大鼠早期腎髒損害.
목적 탐토백려호순대당뇨병대서신피질단백연선알선표령이핵감산린산양화매4(NOX4)、포도당조절단백78( GRP78)표체적영향,이연구백려호순대당뇨병대서신피질양화응격손상적보호작용.방법 장15지SD웅성대서응용련뇨좌균소(STZ)제조당뇨병대서모형.13주후장조모성공적12지대서수궤분위당뇨병조(DM조,n=6)화백려호순간예조(DR,n=6),이6지건강SD대서작위대조조(NC조).DR조대서급여백려호순10 mg·kg-1·d-1매일고정시간련속관위2주,DM조용등체적0.5％최갑기섬유소납용액관위,지제14주결속.제14주결속시,검측3조대서적혈당、체중、혈기항、혈뇨소담、24 h뇨미량백단백.취신조직,제석사절편주PAS염색관찰신소구신소관형태학변화.취10％신피질조직균장후측정병이철(MDA)、초양화물기화매(SOD)、과양화경매(CAT)수평.Western blotting법검측신피질NOX4화GRP78단백표체적개변.결과 여NC조상비,DM조대서혈당、혈기항、혈뇨소담、24 h뇨미량백단백균현저승고(t=- 52.324、- 20.487、- 20.724、- 55.476,균P＜0.0167),이체중칙명현하강(t=5.820,P ＜0.0167)；백려호순간예후,DR조대서24 h뇨미량백단백、혈기항、혈뇨소담균비DM조정호전추세(t=13.963、7.849、8.678,균P＜0.0167)；이DR조체중화혈당여DM조상비차이무통계학의의(t=-1.767、1.876,균P＞0.0167).여NC조상비,DM조MDA수평명현증가(t=- 10.661,P＜0.0167),이SOD、CAT수평강저(t=8.124、8.222,균P＜0.0167)；이여DM조상비,DR조SOD수평승고(t=- 12.309,p＜0.0167),MDA수평강저(t=4.475,P＜0.0167),2조CAT활력차이무통계학의의(t=-3.029,P＞0.0167).신장조직PAS염색현시NC조대서기저막완정무증후,DM조대서신소구계막기질증다,계막세포증생,DR조칙교DM조감경.Western blotting현시DM조혈관조직중NOX4급GRP78단백표체상조(0.49±0.05、1.47±0.04),균고우대조조NC조(0.09±0.001、0.84±0.012,t=- 14.255、-25.179,균P＜ 0.0167)；DR조NOX4단백표체(0.30 ±0.073)잉고우NC조(t=-5.125,P＜0.0167)；GRP78단백(0.60 ±0.034)칙저우NC조(t=- 28.017,P＜0.0167).결론 당뇨병대서신피질상존재착명현적양화응격손상,백려호순가능통과억제GRP78단백적표체종이영향내질망응격,억제NOX4단백적표체감경양화응격대당뇨병대서조기신장손해.
Objective To investigate the effect of resveratrol (Res)on the protein expression of reduced form of nicotinamide-adenine dinucleotide phosphate oxidase 4 (NOX4) and glucose-regulated protein 78 (GRP78) and its protective effects on the oxidative stress injuries in the renal cortex of diabetic rats.Methods Fifteen SD rats were given a single intraperitoneal injection of streptozotocin(STZ) to set up animal models of diabetes.Twelve weeks after STZ injection,12 rat diabetes models were established successfully and were randomized into diabetes mellitus group ( group DM,n =6 ) and Res intervention group ( group DR,n =6).Another 6 untreated healthy SD rats served as norml control (group NC).Rats in DR group were administcrcd intragastricly with Res 10 mg·kg-1·d-1 while those in DM group with equal volume of 0.5％ sodium carboxymethylcellulose regularly for 2 weeks.At the end of the experiments,the blood glucose (BG),body weight (BW),serum creatinine (Scr),blood urea nitrogen (BUN),24 urinary albumin excretion(UAE) of all rats were measured.Renal cortex from the groups were embedded in paraffin and sectioned for morphological studies. The activity of malondialdehyde (MDA),superoxide dismutase (SOD) and catalase (CAT) in the renal cortex was assayed by chromatometry.The expression of NOX4 and GRP78 proteins in the renal cortex were detected by using Western blotting.Results Compared with those in group NC,the BW in group DM decreased,while BG,Scr,BCN and 24h UAE increased significantly (t=- 52.324, - 20.487, - 20.724, - 55.476,all P ＜ 0.0167). It indicated that resveratrol improved the 24 h UAE,Ser and BUN in group DR as compared with those in group DM ( t =13.963,7.849,8.678,all P ＜0.0167); but there was no significant differences in BG and BW between the two groups( t =- 1.767,1.876,all P ＞ 0.0167).Moreover,the MDA activity in DM group increased while the SOD and CAT activity decreased significantly as compared with those in group NC( t =- 10.661,8.124,8.222,all P ＜ 0.0167).The SOD activity was significantly higher while MDA was significantly lower in group DR than those in DM group (t =- 12.309,4.475,all P ＜0.0167); no significant differences in CAT activity was observed between the two groups (t =-3.029,P ＞ 0.0167).The glomerular basement membrane was intact in NC group,more glomerular mesangial matrix and cell proliferation was observed in group DM and which were attenuated in the group DR.The expression of NOX4 and GRP78 in the blood vessels of group DM were up-regulated than those in group NC ( t =- 14.255,- 25.179,P ＜0.0167).After the administration of resveratrol,the expression of NOX4 was still up-regulated while GRP48was down-regulated in group DR as compared with those in group NC (t =- 5.125, - 28.017,P ＜0.0167).Conclusions Oxidative stress is present in the renal cortex of dialetic rats.It suggests that resveratrol protect the renal cortex of diabetic rats by suppressing the expression of NOX4 and GRP78 to attenuate endoplasmic reticulum stress and subsequent oxidative stress injuries during the early stage of diabetes.
