目的 评价蛋白激酶Cα(PKCα)在电针减轻内毒素休克诱发兔急性肾损伤中的作用及其与核因子E2相关因子2(Nrf2)/血红素加氧酶-1(HO-1)通路的关系.方法 健康清洁级雄性新西兰大白兔80只,2月龄,体重1.5 ～ 2.0 kg,采用随机数字表法分为8组(n=10):假手术组(S组)、内毒素休克致急性肾损伤组(AKI组)、PKCα抑制剂-白屈菜赤碱+AKI组(CHA组)、白屈菜赤碱组(Che组)、溶媒-二甲基亚砜组(D组)、电针刺激穴位+AKI组(EA组)、电针刺激非穴位+AKI组(SEA组)和电针刺激穴位+白屈菜赤碱+AKI组(CEA组).EA组和CEA组于模型制备前1-4 d及模型制备过程中电针刺激双侧足三里和肾俞穴,疏密波,频率2/15 Hz,刺激电流1～2 mA,波宽0.2～0.6 ms,刺激强度以兔肢体出现轻微肌颤为宜,1次/d,30 min/次.SEA组以同样方法电针刺激足三里和肾俞穴旁开0.5 cm非经非穴处.AKI组、CHA组、EA组、SEA组和CEA组采用耳缘静脉注射脂多糖5 mg/kg的方法制备内毒素休克致急性肾损伤模型,S组、Che组和D组给予等容量生理盐水.于模型制备前30 min,CHA组和CEA组静脉注射白屈菜赤碱5 mg/kg(溶于0.5 ml 0.1％二甲基亚砜),Che组给予等量白屈菜赤碱,D组给予二甲基亚砜0.5 ml.静脉注射脂多糖或生理盐水后6h时,采集颈内动脉血样,检测血清BUN和Cr浓度,处死大鼠后取肾组织,行病理学观察并进行评分,测定肾组织MDA含量及SOD活性,检测肾组织PKCα、HO-1蛋白、核蛋白及总蛋白中Nrf2的表达水平.结果 与S组比较,AKI组、CHA组、EA组、SEA组和CEA组血清BUN和Cr浓度升高,肾组织MDA含量升高,SOD活性降低,肾组织病理学评分升高,PKCα、HO-1蛋白、核蛋白及总蛋白Nrf2的表达上调(P＜0.05);与AKI组比较,EA组血清BUN和Cr浓度降低,肾组织MDA含量降低,SOD活性升高,肾组织病理学评分降低,PKCα、HO-1蛋白、核蛋白及总蛋白Nrf2的表达上调,CHA组和CEA组血清BUN和Cr浓度升高,肾组织MDA含量升高,SOD活性降低,肾组织病理学评分升高,PKCα、HO-1蛋白、核蛋白及总蛋白Nrf2的表达下调(P＜0.05);与EA组比较,CEA组血清BUN和Cr浓度升高,肾组织MDA含量升高,SOD活性降低,肾组织病理学评分升高,PKCα、HO-1蛋白、核蛋白及总蛋白Nrf2的表达下调(P＜0.05);与CEA组比较,CHA组血清BUN和Cr浓度升高,肾组织MDA含量升高,SOD活性降低,肾组织病理学评分升高,PKCα、HO-1蛋白、核蛋白及总蛋白Nrf2的表达下调(P＜0.05).结论 PKCα介导电针足三里和肾俞穴减轻内毒素休克诱发兔急性肾损伤的作用,其机制可能与激活Nrf2/HO-1通路有关.
目的 評價蛋白激酶Cα(PKCα)在電針減輕內毒素休剋誘髮兔急性腎損傷中的作用及其與覈因子E2相關因子2(Nrf2)/血紅素加氧酶-1(HO-1)通路的關繫.方法 健康清潔級雄性新西蘭大白兔80隻,2月齡,體重1.5 ～ 2.0 kg,採用隨機數字錶法分為8組(n=10):假手術組(S組)、內毒素休剋緻急性腎損傷組(AKI組)、PKCα抑製劑-白屈菜赤堿+AKI組(CHA組)、白屈菜赤堿組(Che組)、溶媒-二甲基亞砜組(D組)、電針刺激穴位+AKI組(EA組)、電針刺激非穴位+AKI組(SEA組)和電針刺激穴位+白屈菜赤堿+AKI組(CEA組).EA組和CEA組于模型製備前1-4 d及模型製備過程中電針刺激雙側足三裏和腎俞穴,疏密波,頻率2/15 Hz,刺激電流1～2 mA,波寬0.2～0.6 ms,刺激彊度以兔肢體齣現輕微肌顫為宜,1次/d,30 min/次.SEA組以同樣方法電針刺激足三裏和腎俞穴徬開0.5 cm非經非穴處.AKI組、CHA組、EA組、SEA組和CEA組採用耳緣靜脈註射脂多糖5 mg/kg的方法製備內毒素休剋緻急性腎損傷模型,S組、Che組和D組給予等容量生理鹽水.于模型製備前30 min,CHA組和CEA組靜脈註射白屈菜赤堿5 mg/kg(溶于0.5 ml 0.1％二甲基亞砜),Che組給予等量白屈菜赤堿,D組給予二甲基亞砜0.5 ml.靜脈註射脂多糖或生理鹽水後6h時,採集頸內動脈血樣,檢測血清BUN和Cr濃度,處死大鼠後取腎組織,行病理學觀察併進行評分,測定腎組織MDA含量及SOD活性,檢測腎組織PKCα、HO-1蛋白、覈蛋白及總蛋白中Nrf2的錶達水平.結果 與S組比較,AKI組、CHA組、EA組、SEA組和CEA組血清BUN和Cr濃度升高,腎組織MDA含量升高,SOD活性降低,腎組織病理學評分升高,PKCα、HO-1蛋白、覈蛋白及總蛋白Nrf2的錶達上調(P＜0.05);與AKI組比較,EA組血清BUN和Cr濃度降低,腎組織MDA含量降低,SOD活性升高,腎組織病理學評分降低,PKCα、HO-1蛋白、覈蛋白及總蛋白Nrf2的錶達上調,CHA組和CEA組血清BUN和Cr濃度升高,腎組織MDA含量升高,SOD活性降低,腎組織病理學評分升高,PKCα、HO-1蛋白、覈蛋白及總蛋白Nrf2的錶達下調(P＜0.05);與EA組比較,CEA組血清BUN和Cr濃度升高,腎組織MDA含量升高,SOD活性降低,腎組織病理學評分升高,PKCα、HO-1蛋白、覈蛋白及總蛋白Nrf2的錶達下調(P＜0.05);與CEA組比較,CHA組血清BUN和Cr濃度升高,腎組織MDA含量升高,SOD活性降低,腎組織病理學評分升高,PKCα、HO-1蛋白、覈蛋白及總蛋白Nrf2的錶達下調(P＜0.05).結論 PKCα介導電針足三裏和腎俞穴減輕內毒素休剋誘髮兔急性腎損傷的作用,其機製可能與激活Nrf2/HO-1通路有關.
목적 평개단백격매Cα(PKCα)재전침감경내독소휴극유발토급성신손상중적작용급기여핵인자E2상관인자2(Nrf2)/혈홍소가양매-1(HO-1)통로적관계.방법 건강청길급웅성신서란대백토80지,2월령,체중1.5 ～ 2.0 kg,채용수궤수자표법분위8조(n=10):가수술조(S조)、내독소휴극치급성신손상조(AKI조)、PKCα억제제-백굴채적감+AKI조(CHA조)、백굴채적감조(Che조)、용매-이갑기아풍조(D조)、전침자격혈위+AKI조(EA조)、전침자격비혈위+AKI조(SEA조)화전침자격혈위+백굴채적감+AKI조(CEA조).EA조화CEA조우모형제비전1-4 d급모형제비과정중전침자격쌍측족삼리화신유혈,소밀파,빈솔2/15 Hz,자격전류1～2 mA,파관0.2～0.6 ms,자격강도이토지체출현경미기전위의,1차/d,30 min/차.SEA조이동양방법전침자격족삼리화신유혈방개0.5 cm비경비혈처.AKI조、CHA조、EA조、SEA조화CEA조채용이연정맥주사지다당5 mg/kg적방법제비내독소휴극치급성신손상모형,S조、Che조화D조급여등용량생리염수.우모형제비전30 min,CHA조화CEA조정맥주사백굴채적감5 mg/kg(용우0.5 ml 0.1％이갑기아풍),Che조급여등량백굴채적감,D조급여이갑기아풍0.5 ml.정맥주사지다당혹생리염수후6h시,채집경내동맥혈양,검측혈청BUN화Cr농도,처사대서후취신조직,행병이학관찰병진행평분,측정신조직MDA함량급SOD활성,검측신조직PKCα、HO-1단백、핵단백급총단백중Nrf2적표체수평.결과 여S조비교,AKI조、CHA조、EA조、SEA조화CEA조혈청BUN화Cr농도승고,신조직MDA함량승고,SOD활성강저,신조직병이학평분승고,PKCα、HO-1단백、핵단백급총단백Nrf2적표체상조(P＜0.05);여AKI조비교,EA조혈청BUN화Cr농도강저,신조직MDA함량강저,SOD활성승고,신조직병이학평분강저,PKCα、HO-1단백、핵단백급총단백Nrf2적표체상조,CHA조화CEA조혈청BUN화Cr농도승고,신조직MDA함량승고,SOD활성강저,신조직병이학평분승고,PKCα、HO-1단백、핵단백급총단백Nrf2적표체하조(P＜0.05);여EA조비교,CEA조혈청BUN화Cr농도승고,신조직MDA함량승고,SOD활성강저,신조직병이학평분승고,PKCα、HO-1단백、핵단백급총단백Nrf2적표체하조(P＜0.05);여CEA조비교,CHA조혈청BUN화Cr농도승고,신조직MDA함량승고,SOD활성강저,신조직병이학평분승고,PKCα、HO-1단백、핵단백급총단백Nrf2적표체하조(P＜0.05).결론 PKCα개도전침족삼리화신유혈감경내독소휴극유발토급성신손상적작용,기궤제가능여격활Nrf2/HO-1통로유관.
Objective To evaluate the role of protein kinase Ca (PKCα) in electroacupuncture (EA)-induced reduction of acute kidney injury (AKI) induced by endotoxic shock,and the relationship with nuclear factor E2-related factor 2/heme oxygenase-1 Nrf2/HO-1 pathway in rabbits.Methods Eighty heahhy male New Zealand white rabbits,aged 2 months,weighing 1.5-2.0 kg,were randomly divided into 8 groups (n=10 each) using a random number table:sham operation group (group S),group AKI,specific PKCα inhibitor chelerythrine + AKI group (group CHA),chelerythrine group (group Che),dimethyl sulfoxide (DMSO) group (group D),EA at acupoints + AKI group (group EA),EA at non-acupoints + AKI group (group SEA),and EA at acupoints + chelerythrine + AKI group (group CEA).Bilateral 30 min EA (disperse-dense wave,wave length 0.2-0.6 ms,frequency 2/15 Hz,intensity 1-2 mA) stimulation of Zusanli and Shenshu acupoints was performed once a day for 4 days before establishment of the model and during the process of establishment of the model in EA and CEA groups.In group SEA,EA was performed at the points 0.5 cm lateral to the acupoints of Zusanli and Shenshu with the same parameters.The animals were anesthetized with iv 20％ urethane 5 ml/kg,tracheostomized and kept spontaneous breathing.Lipopolysaccharide 5 mg/kg (in 2 ml of normal saline) was injected via the auricular vein to establish the model of endotoxic shock-induced AKI in AKI,CHA,EA,SEA and CEA groups,while the equal volume of normal saline was given in S,Che and D groups.At 30 min before establishment of the model,chelerythrine 5 mg/kg (in 0.5 ml of 1％ DMSO) was injected intravenously in CHA and CEA groups,the equal volume of chelerythrine was given in Che group,while the equal volume of DMSO was given in group D.At 6 h after lipopolysaccharide or normal saline injection,blood samples were taken from the internal carotid artery for determination of serum urea nitrogen (BUN) and creatinine (Cr) concentrations.The rabbits were then sacrificed by exsanguinations.The kidney specimens were removed for microscopic examination of pathologic changes which were scored and for determination of superoxide dismutase (SOD) activities,malondialdehyde (MDA) contents,and expression of PKCα protein and HO-1 protein,and expression of Nrf2 in nucleoprotein and total protein.Results Compared with group S,the serum BUN and Cr concentrations were significantly increased,MDA contents were increased,the activities of SOD were decreased,the kidney injury scores were increased,and the expression of PKCα protein,HO-1 protein,and Nrf2 in nucleoprotein and total protein was up-regulated in AKI,CHA,EA,SEA and CEA groups.Compared with group AKI,the serum BUN and Cr concentrations were significantly decreased,MDA contents were decreased,the activities of SOD were increased,the kidney injury scores were decreased,and the expression of PKCα protein,HO-1 protein,and Nrf2 in nucleoprotein and total protein was up-regulated in group EA,and the serum BUN and Cr concentrations were significantly increased,MDA contents were increased,the activities of SOD were decreased,the kidney injury scores were increased,and the expression of PKCα protein,HO-1 protein,and Nrf2 in nucleoprotein and total protein was down-regulated in CHA and CEA groups.The serum BUN and Cr concentrations were significantly higher,MDA contents were higher,the activities of SOD were lower,the kidney injury scores were higher,and the expression of PKCα protein,HO-1 protein,and Nrf2 in nucleoprotein and total protein was lower in group CEA than in group EA,and in CHA group than in CEA group.Conclusion PKCα mediates reduction of endotoxic shock-induced AKI by EA of Zusanli and Shenshu acupoints in rabbits,and the mechanism may be related to activation of Nrf2/HO-1.
