The Expression Changes Of Dipeptidyl Peptidase Ⅲ In D-galactose Cataractous Rat Lens

Cataract is one type of global blind eyes disease. Especially, it occurs frequently among the middle aged and even older group of people. History of study on cataract has already been up to more than 100 years in ophthalmology. However, the reason which causes the disease has not been reached common understanding. Nowadays, the topic of interest in studying is about the change of the crystallin influencing the forming of cataract. A lot of domestic and international experts demonstrated the crystallin turn from solubility into insolubility. This kind of irreversible change mainly causes the forming of cataract, which is directly influenced by the protein abnormal catabolism. But the concrete mechanism of this kind of pathological changes is not very clear. It is thought that the crystallin may be degraded by some protein hydrolase, which make the crystallin lose normal structure and physiological function or turn from soluble protein into a unsolvable protein. Thus the cataract is formed.Dipeptidyl peptidases (DPPs) are a group of peptidases thatcleave dipeptides from the N-terminus of peptide substrates. They have been identified in various mammalian tissues. All DPPs are classified into different types according to subcellular location, substrate specificity and inhibitory sensitivity.Dipeptidyl peptidase III(DPP III), discovered by Ellis and Nuenke in the bovine anterior pituitary[29], DPPIII was purifies by the cytosolic, which is a zinc metallo-exopeptidase whose active center is primarily serine. The calculated molecular weight of the purified enzyme was 82845.6 according to TOF-MS, and 82000 on non-denatured PAGE and 82000 on SDS-PAGE in the absence or presence of beta-ME[30].DPPIII rapidly hydrolyzed the substrate cleaves Arg-Arg- b-naphthylamide in a pH range of 7. 5 to 9.5, is inhibited by thiol reagents. The enzyme from rat brain cytosol prefers angiotensins and enkephalins as substrates at pH 7.4 and is inhibited not only by thiol reagents but also by the metalchelating agent o-phenanthroline [31]. DPP III has been demonstrated to be a metalloenzyme by inhibition and restoration experiments on the activity utilizing metal chelators and metal ions [32]. However, humanity's cataractous lens [33] and pig's spleen DPPIII could still beinhibited by di-isopropyl-uorophosphate (DFP) and the metal-chelating agents. The DPPIII metallic ion is the active position spot, and directly affects the response between DPPIII and the substrate. And Co(2+), Ni(2+) and Cu(2+) may replace Zn(2+) as well, which causes the complex of deactivation DPPIII [35].Some research proved [36]that DPPIII has the different active change in the mouse's different organization, including brain, heart, lung, liver, small intestine, red blood cell. And proved that, in the identical big mouse, in the relative high-oxygen organization (e.g. lung, heart and red blood cell), the DPPIII activeness must be lower[36]. DPPIII widely exists in human's embryo, the crystalline lens, the red blood cell, the seminal fluid [37, 33, 38] as a protein hydroltyic enzyme. Recently, some research proves the DPPIII active change existing in the Drosophila melanogaster, adult schistosomes [39,40].Purpose: This research is made by establishing cataract rat model by injecting the D- galactose to abdominal cavity, and examining the changes in lens of cataractous rat by using the Westen-blotting to examine DPPIII. We may discuss its relativity to cataract morbidity.Methods:(1) Establishing the D- galactose cataractous rat model, namely age relevant cataractous rat model. Choosing 5~6 week age Wistar rat, female and male, weight 55±5g, altogether 60.After dripping the compound tropicamide drop to disperse the pupil of the rats, we may inspect the lens transparency under the slit lamp. Then we may divide the rats into the normal comparison group (20 ) and the D- galactose cataractous model group, namely the experimental group (40).The experimental group rats were injected D- galactose in abdominal cavity (30g/kg/d),2/d, continual seven to twenty-eight days; Normal comparison group rats were injected isometric 0.9% asepsis physiological saline in abdominal cavity at the same time, 2/d, continual seven to twenty-eight days.During the period of modeling, After dripping the compound tropicamide drop to disperse the pupil of the experimental group of rats, we may observe the bleary situation of lens. According to nature and the degree of bleary situation, we may divide them into I period (The cyst period): after one week injection; II period (cperiod period): after two weeks injection; III period(Mature period): after three weeks injection; IV period (the ripe period) four weeks after injection;. In theexperimental group, the rats whose lens appeared I, II period of change were the mild cataract group(test 1 group) ; The rats whose lens appear III, IV period of change were the serious cataract group. At the same time, we may divides the normal control group into control group one and control group two.(2) We may examine changes of DPPIII in rats'lens between the normal control group and the experimental group by using Western Blotting (immunity signature) method.Results:1.The D- galactose cataractous rats model was successfully established, and carried on correspondingly division of stages and groups.2.Western Blotting method examination results:(1) The DPPIII expression of lens in experimental groups is higher than the corresponding period control grouping. Namely the DPPIII expression of lens in the first experimental groups is higher than the first control group; the DPPIII expression of lens in the second experimental groups is higher than the sedond control group.(2) In experimental groups, the DPPIII expression of lens in the second experimental groups is higher than the first experimental group.(3) In control groups, the DPPIII expression of lens in the second control group is higher than the first control group. Conclusions:(1) Using the D- galactose injection to abdominal cavity can succeed in establishing cataractous rat model. Its lens bleary degree is similar to old-aged cataract. (2) The normal rats'lens has DPPIII, its expression increases along the advancing of the rats'age. They correlate with each other.(3) The DPPIII expression of lens in cataractous rats is higher than the comparison group. Moreover, the DPPIII expression and lens bleary degree are correlated.Based on these results, this experiment demonstrated that DPPIII may have the vital function in the formation and the aggravation of cataract.