中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2011年
10期
894-898
,共5页
董志军%陶相宜%郭立涛%张铁民%王海彬%付笑笑
董誌軍%陶相宜%郭立濤%張鐵民%王海彬%付笑笑
동지군%도상의%곽립도%장철민%왕해빈%부소소
中药/菩人丹超微粉%视网膜/糖尿病视网膜病变%细胞凋亡%线粒体
中藥/菩人丹超微粉%視網膜/糖尿病視網膜病變%細胞凋亡%線粒體
중약/보인단초미분%시망막/당뇨병시망막병변%세포조망%선립체
Traditional Chinese medicion/purendan supermicropowder%Retina/diabetic retinopathy%cell apoptosis%Mitochondria
背景 研究表明线粒体途径在细胞的凋亡过程中发挥重要作用,而菩人丹(PRD)超微粉对视网膜神经细胞的凋亡可能有保护作用.目的 探讨PRD对糖尿病大鼠视网膜醛糖还原酶(AR)活性、神经细胞凋亡及线粒体凋亡途径的影响.方法 采用随机数字表法将36只清洁级Wistar大鼠分为正常对照组、糖尿病模型组及PRD治疗组,每组12只.糖尿病模型组、PRD治疗组大鼠均采用链脲佐菌素(STZ)25mg/(kg·d)连续腹腔注射3d,每日1次,建立2型糖尿病大鼠模型,以血糖≥16.7 mmoL/L为造模成功.模型成功建立后,PRD治疗组大鼠给予1.8g/(kg·d)PRD灌胃,每日1次,连续3个月.给药结束后取大鼠眼球,分离视网膜组织并制备匀浆和切片,采用紫外分光光度法检测视网膜组织中的AR活性；采用TUNEL 法检测大鼠视网膜神经细胞的凋亡并计算凋亡指数(AI)；用Western blot法检测视网膜组织中B细胞淋巴瘤/白血病-2(bcl-2)、bcl-2相关X蛋白(bax)、细胞色素C(cyt-c)及半胱天冬酶-3(caspase-3)蛋白的表达.结果 正常对照组、糖尿病模型组和PRD治疗组大鼠视网膜组织中AR活性和AI的总体比较差异均有统计学意义(F=90.115、165.540,P＜0.01),其中糖尿病模型组大鼠视网膜组织中的AR活性和神经细胞AI均明显高于正常对照组和PRD治疗组,差异均有统计学意义(P＜0.01),TUNEL染色示阳性细胞主要位于视网膜神经节细胞(RGC)层和内核层.正常对照组、糖尿病模型组和PRD治疗组大鼠视网膜组织中bax、cyt-c、caspase-3、bcl-2蛋白表达及bcl-2/bax值的表达明显不同,差异均有统计学意义(F=51.332、41.262、25.888、38.564、47.870,P＜0.01),其中糖尿病模型组明显高于正常对照组和PRD治疗组,但bcl-2蛋白表达、bcl-2/bax比值则明显低于正常对照组和PRD治疗组,差异均有统计学意义(P＜0.01).大鼠视网膜AR活性、神经细胞AI、bax、cyt-c及caspase-3蛋白表达PRD治疗组与糖尿病模型组比较均明显降低,bcl-2蛋白表达、bcl-2/bax值则显著升高,差异均有统计学意义(P＜0.01).结论 PRD可通过抑制AR活性、上调凋亡抑制基因bcl-2的表达及下调凋亡促进基因bax、cyt-c和caspase-3的表达,抑制线粒体凋亡途径活化,减少糖尿病大鼠视网膜神经细胞的凋亡,发挥对糖尿病视网膜损伤的保护作用.
揹景 研究錶明線粒體途徑在細胞的凋亡過程中髮揮重要作用,而菩人丹(PRD)超微粉對視網膜神經細胞的凋亡可能有保護作用.目的 探討PRD對糖尿病大鼠視網膜醛糖還原酶(AR)活性、神經細胞凋亡及線粒體凋亡途徑的影響.方法 採用隨機數字錶法將36隻清潔級Wistar大鼠分為正常對照組、糖尿病模型組及PRD治療組,每組12隻.糖尿病模型組、PRD治療組大鼠均採用鏈脲佐菌素(STZ)25mg/(kg·d)連續腹腔註射3d,每日1次,建立2型糖尿病大鼠模型,以血糖≥16.7 mmoL/L為造模成功.模型成功建立後,PRD治療組大鼠給予1.8g/(kg·d)PRD灌胃,每日1次,連續3箇月.給藥結束後取大鼠眼毬,分離視網膜組織併製備勻漿和切片,採用紫外分光光度法檢測視網膜組織中的AR活性；採用TUNEL 法檢測大鼠視網膜神經細胞的凋亡併計算凋亡指數(AI)；用Western blot法檢測視網膜組織中B細胞淋巴瘤/白血病-2(bcl-2)、bcl-2相關X蛋白(bax)、細胞色素C(cyt-c)及半胱天鼕酶-3(caspase-3)蛋白的錶達.結果 正常對照組、糖尿病模型組和PRD治療組大鼠視網膜組織中AR活性和AI的總體比較差異均有統計學意義(F=90.115、165.540,P＜0.01),其中糖尿病模型組大鼠視網膜組織中的AR活性和神經細胞AI均明顯高于正常對照組和PRD治療組,差異均有統計學意義(P＜0.01),TUNEL染色示暘性細胞主要位于視網膜神經節細胞(RGC)層和內覈層.正常對照組、糖尿病模型組和PRD治療組大鼠視網膜組織中bax、cyt-c、caspase-3、bcl-2蛋白錶達及bcl-2/bax值的錶達明顯不同,差異均有統計學意義(F=51.332、41.262、25.888、38.564、47.870,P＜0.01),其中糖尿病模型組明顯高于正常對照組和PRD治療組,但bcl-2蛋白錶達、bcl-2/bax比值則明顯低于正常對照組和PRD治療組,差異均有統計學意義(P＜0.01).大鼠視網膜AR活性、神經細胞AI、bax、cyt-c及caspase-3蛋白錶達PRD治療組與糖尿病模型組比較均明顯降低,bcl-2蛋白錶達、bcl-2/bax值則顯著升高,差異均有統計學意義(P＜0.01).結論 PRD可通過抑製AR活性、上調凋亡抑製基因bcl-2的錶達及下調凋亡促進基因bax、cyt-c和caspase-3的錶達,抑製線粒體凋亡途徑活化,減少糖尿病大鼠視網膜神經細胞的凋亡,髮揮對糖尿病視網膜損傷的保護作用.
배경 연구표명선립체도경재세포적조망과정중발휘중요작용,이보인단(PRD)초미분대시망막신경세포적조망가능유보호작용.목적 탐토PRD대당뇨병대서시망막철당환원매(AR)활성、신경세포조망급선립체조망도경적영향.방법 채용수궤수자표법장36지청길급Wistar대서분위정상대조조、당뇨병모형조급PRD치료조,매조12지.당뇨병모형조、PRD치료조대서균채용련뇨좌균소(STZ)25mg/(kg·d)련속복강주사3d,매일1차,건립2형당뇨병대서모형,이혈당≥16.7 mmoL/L위조모성공.모형성공건립후,PRD치료조대서급여1.8g/(kg·d)PRD관위,매일1차,련속3개월.급약결속후취대서안구,분리시망막조직병제비균장화절편,채용자외분광광도법검측시망막조직중적AR활성；채용TUNEL 법검측대서시망막신경세포적조망병계산조망지수(AI)；용Western blot법검측시망막조직중B세포림파류/백혈병-2(bcl-2)、bcl-2상관X단백(bax)、세포색소C(cyt-c)급반광천동매-3(caspase-3)단백적표체.결과 정상대조조、당뇨병모형조화PRD치료조대서시망막조직중AR활성화AI적총체비교차이균유통계학의의(F=90.115、165.540,P＜0.01),기중당뇨병모형조대서시망막조직중적AR활성화신경세포AI균명현고우정상대조조화PRD치료조,차이균유통계학의의(P＜0.01),TUNEL염색시양성세포주요위우시망막신경절세포(RGC)층화내핵층.정상대조조、당뇨병모형조화PRD치료조대서시망막조직중bax、cyt-c、caspase-3、bcl-2단백표체급bcl-2/bax치적표체명현불동,차이균유통계학의의(F=51.332、41.262、25.888、38.564、47.870,P＜0.01),기중당뇨병모형조명현고우정상대조조화PRD치료조,단bcl-2단백표체、bcl-2/bax비치칙명현저우정상대조조화PRD치료조,차이균유통계학의의(P＜0.01).대서시망막AR활성、신경세포AI、bax、cyt-c급caspase-3단백표체PRD치료조여당뇨병모형조비교균명현강저,bcl-2단백표체、bcl-2/bax치칙현저승고,차이균유통계학의의(P＜0.01).결론 PRD가통과억제AR활성、상조조망억제기인bcl-2적표체급하조조망촉진기인bax、cyt-c화caspase-3적표체,억제선립체조망도경활화,감소당뇨병대서시망막신경세포적조망,발휘대당뇨병시망막손상적보호작용.
Background Research demonstrated that mitochondrial pathway plays a key role in cell apoptosis.Purendan supermicropowder(PRD),a traditional Chinese medicine,may be a potentially effective therapy for neuron apoptosis in diabetic retina.Objective This study was carried out to investigate the effects of PRD on aldose reductase(AR)activity,neuron apoptosis and mitochondrial pathway in retina of diabetic rat.Methods Thirty-six clean male Wistar rats were randomly divided into normal control group,diabetes model group,PRD treatment group randomly and 12 rats for each group.The diabetes models were established by intraperitoneal injection of 25 mg/(kg · d)streptozotocin(STZ)for 3 consecutive days,and blood glucose ≥ 16.7 mmol/L was taken as the standard.PRD solution of 1.8 g/(kg · d)was lavaged in 12 models for 3 months.The eyeballs were enucleated for the preparation of retinal tissue homogenate and slice.AR activity in the retina was detected by ultraviolet spectrophotometry,and neuron apoptosis in retina was assayed by TUNEL staining.Western blot was used to assess the expressions of bcl-2,bax,cyt-c and caspase-3 protein in the retina.The use of animals followed the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Committee(Version 1988).Results Statistically significant differences were found in AR activity and AI among the normal control group,diabetic group and PRD groups(F=90.115,165.540,P＜0.01),and those of diabetic group were evidently higher than the normal control group and PRD group(P＜0.01,P＜0.01).The positive TUNEL cells mainly located in inner nuclear layer and retinal ganglion cell layer.The expressions of bax,cyt-c,caspase-3,bcl-2 and bcl-2/bax in retina were obviously different among these three groups(F =51.332,41.262,25.888,38.564,47.870,P＜0.01),and the expression of bax,cyt-c and caspase-3 protein in diabetic group evidently elevated in comparison with the normal control group and PRD group(t ＝ 10.32,11.04,6.91,P ＜ 0.01)and the expressions of bcl-2 protein and bcl-2/bax value were significantly lower in diabetic rats than in the normal control rats(t =18.05,12.23,P＜0.01).AR activity by AI of retina,the expressions of bax,cyt-c and caspase-3 proteins in retina were obviously lower in PRD group than in diabetes model rats(P ＜ 0.01),and the expression of bcl-2 protein and bcl-2/bax value were significantly higher in PRD group than in diabetes group(P＜0.01).Conclusions PRD can protect retina against the damage caused by high glucose by suppressing AR activity by downregulating the expressions of bax,cyt-c,caspase-3 proteins,increasing the expressions of bcl-2 protein in retina of diabetic rats and further inhibiting the mitochondrial pathway and reducing cell apoptosis in retina of diabetic rats.
