The Preliminary Study Of Screening The Active Substance From Dendrobium. With The Function Of Preventing Sugar Cataract And Screening Its Target Proteins

AIM:To screen out an active substance from Dendrobium.on preventing sugar cataract and to find out its target proteins,then to provide a basic theory for exploiting Dendrobium.praeparatum with the effect of anti-sugar cataract.METHODS:1.The total alkaloids and polysaccharides were extracted from dendrobium nobile Lindl.by the chemical-dissolvent method;35 crystalline lenses of Wistar rat were divided into 7 groups at random:normal control group(DMEM medium+10%fetal calf serum +1%penicilin and streptomycin solution),model group(the medium as normal control group +2mmol/L30%H₂O₂),positive control group(the medium as model group +10%Pirenoxine sodium eye drops),high dose(or low dose)total alkaloids group(the medium as model group +2mmoL/L30%H₂O₂+4mg/mLor 2mg/mL total alkaloids or polysaccharides),high dose(or low dose)polysaccharides(the medium as model group +2mmoL/L30%H₂O₂+4mg/mLor 2mg/mL polysaccharides),all lenses were cultured in 5%CO₂,37.0℃incubator.At the time of 6h,12h,18h,24h after culturing,the opacity of each crystalline was observed with an anatomical microscope.At last,all crystallines were embedded in paraffin,stained with hematoxyin and eosin(H&E),the level of water-solubility protein,GSH,MDA and the activity of T-SOD of each lens was measured respectively.2.4～5 weeks old Wistar-rats were peritoneal injected with D-galactose to produce sugar cataract,and divided into 7 groups at random:the normal control group treated with normal feed,model group(treated with intragastric administration of NS),positive control group(treated with intragastric administration of 0.22g/kg/d Shihuyeguangwan),high or low dose total alkaloids(or polysaccharides)group(treated with intragastric administration of 0.36g/kg/d or 0.18g/kg/d total alkaloids),high or low dose polysaccharides group(treated with intragastric administration of 0.94g/kg/d or 0.47g/kg/d polysaccharides).Lens opacity was observed every 7 days by the slit-lamp microscope. After 6 weeks,total protein of each lens was extracted,proteins with differential expression and target proteins were identifited by using the proteomic technologies such as 2-DE,image analysis,MALDI-TOF-TOF-MS,and bioinformatics analysis.RESULTS:1.Lens opacity could be lightened obviously by the total alkaloids or polysaccharides either,and the pathologic damages were lightened by the two substances too,especially by the total alkaloids;furthermore,the level of water-solubility protein,GSH,and the activity of T-SOD in the lens were up-regulate as the contents of MDA was degraded by the two substances(P＜0.05 or P＜0.01),and the effect of high dose total alkaloids was better than the Pirenoxine sodium eye drops。2.Lens opacity in rats with D-galactose cataract could be lightened by the total alkaloids.Proteome research found that,the level ofβA3,βB3,γB-crystallin,actin cytoplasmic 2,Ubiquitin carboxyl-erminal hydrolase isozyme Ll.were decreased in the rats'lenses with D-galactose cataract,and upgraded in the rats' lenses treated with total alkaloids,and the content level ofαB,βA2,βB1,γN-crystallin were just on the opposite, all the lensproteins and enzyme exceeded 2 times up or down regulation and had 99% statistical significance.CONCLUSION:The total alkaloids of Dendrobium has the effect of anti-sugar cataract in rat,its target proteins may beβA3,βB3,γB-crystallin,actin cytoplasmic 2,and Ubiquitin carboxyl-erminal hydrolase isozyme L1,and the key protein is actin cytoplasmic 2.