Effect And Mechanism Of TPCK On SD Rats Lens Opacity

Cataract is still a main disease of growing blind in our country. And it influencedseriously the health of human all of the world at present which pathogenesis is not yet clear.Cataract is a multi-cause disease such as gene mutation, oxidative stress, radiation, age andso on. That reasons lead to lens opacity and further affect the lens transmission andrefraction of light that manifested in the symptoms is blurred vision.Oxidative stress induced by reactive oxygen species (ROS) has long been implicatedin senile cataract formation. ROS molecules are generated in the lens include superoxideanion, hydroxyl radical, and H2O2. Endogenous systems that can generate ROS in the lensinclude NADPH oxidase, mitochondria, and peroxisomes. Two main sources that produceexogenous ROS are UV light and ionic radiation. ROS molecules produced through theseprocesses in the lens are neutralized by antioxidants and oxidation defense systems. Hence,the lens is also equipped with enzyme systems that can repair the damaged molecules1ordegrade the severely damaged ones. Thioredoxin (Trx)/thioredoxin reductase (TR) andthioltransferase (TTase)/glutathione reductase (GR) systems have been identified aspowerful protein repair systems in mammalian tissues. Recent work has shown that bothTTase/GR and Trx/TR systems are present in the lens and play a critical role inmaintaining the lens in a reduced state.Lens proteins (crystallins) is the major structural protein components of the vertebrateeye lens, which are all built in the same manner and contains α-, β-, γ-crystallins accordingto migration in the electricfield. α-crystallins are mainly recognized to function aschaperones in the lens, and γ-crystallins basically maintain the lens chape as a structuralprotein, but little is known about β-crystallins. The regular structure and water-solubleingredient point to an important role for lens high refractive index and transparence thatmakes β-crystallins become a central issue in recent years. There are cysteine in crystallinswhich are extremely sensitive to oxidation due to the thiol. Adjacent thiol sufferingoxidation can form disulfide mixture that is the main reason of crystallins unfolding,aggregation. So lens could maintain transparent relying sufficient amount thiol.Post-translational modification of crystallins is the chief incentives. CrystallinsPTM is more than other protein because its special struture that transparency, lack of bloodsupply, difficult to update. Crystallins water-soluble decline, aggregation enhanced result in high molecular weight which measure up to1×107Da could come cataract accompaniedby light scattering and opacity.Therefor how to keep crystallins free-SH not be cross-linked to disulfide bonds play akey role in maintenance lens transparent and prevention of senile cataract.Preliminary research results in our laboratory show that anti-lenses degeneration ofDTT(C4H10O2S2)is well. It is a chemical reductant that can untie disulfide linkage withinthe protein molecular and intermolecular. It also can inhibit the thermal denaturation oflens protein and non-specificity aggregation as sulfhydryl protective agent. Studies havebeen proved the nucleus plaque of selenite induced rat nuclear cataract after using DTT issignificant less than control group. Our data shows that DTT has obvious inhibitory actionon ultraviolet cataract. DTT can ont only prevent the formation of disulfide bonds betweenprotein molecules but also combine competitively-SH with protein, that can play a role inthe prevention and treatment. Our experiments validated the alkylation of isopropanol oncrystallins thiol so that inhibit the disulfide.Many reagent can take alkylation effect of protein thiol such as tert-butyl alcohol,2-SH-3-butanol and TPCK. Study have been shown that TPCK can inhibit cell apoptosisand anti-inflammtory therefor that have a certain therapeutic effect on cerebral ischemia.Tosyl is a substituent derived from p-toluenesulfonic acid which used as protection ofalcoholic hydroxyl. And methyl as an alkylating can be closed exposed crystallins thiol,inhibit aggregation between proteins, and so it can prevent or delay lens opacity.Thence we cultured lens in vitro and sumulate the environment of oxidative stress.Use TPCK to observe the inhibition of lens opacity, detect enzyme changes and checkapoptosis in order to research effect of TPCK on lens oxidative damage, study itsmechanism.Ubiquitin-proteasome pathway (UPP) is a efficient protein degradation systemdiscovered by Hershko et al. which is related with the pathogenesis and treatment of cancerthat turn into hot content for domestic and foreign academic. Recent study found that UPPis a very complex and careful regulation system of protein degradation which play up-ordown-regulated role by degradation activator or inhibitor factor. Lens epithelial cellscontain all parts of UPP and about40-50%damaged cytosolic protein degradated by UPP.UPP is one of the proteolytic system that selectively degrade modified or damaged proteins which is related to regulation of biological process inside the cells participating lensdifferentiation. Recent years UPP become research hotspot as a important non-lysosomalpathway of intracellular protein degradation but the study about relationship with cataractis still initial stage.This work the last one parts will assess the relationship of cataract and damagedprotein and UPP by means of cultured primary LEC based on latest research results inorder to understand the mechanism of anti-cataract drug and offer clinical evidence. It alsoprovide important theoretical value for other age-related eye disease prevention.Part I The effect of TPCK on lens turbidity and thermal stability under oxidativestressObjective: To investigate the role of TPCK in resistance to oxidative stress andthermal stability in rat lens culture. Method: Intact lenses were cultured in TC-199andthen observe opacity between the oxidative damage group and TPCK group at differenttime points. Detect thermal stability changes of crystallins protein in different groups.Result: The lens in H2O2oxidative damage not acceded TPCK more turbid than the TPCKgroup and the lens homogenates absorbance value lower than TPCK group and thecurvature result is the same trend. Conclusion: TPCK can mitigate lens turbidity underoxidative stress to a certain extent and satbilize the lens protein.Part II The influence of TPCK on lens antioxidant enzymes and apoptosisObjective: To investigate effects of TPCK in antioxidant enzymes and apoptosis inoxidative damaged lens. Method: Cultured rat lens in vitro same as the first part toresearch TTase and Trx contents at different time points and observe apoptosis. Thenculture lens epithelial cells to detect apoptosis after ultraviolet induced. Result: Theactivity of TTase and Trx significant improved after3hours but all reduced rapidly after6hours. The two enzymes in TPCK group are more than the oxidative damaged group butless than the control group. TUNEL results show that lens epithelial cells number is morein the H2O2oxidative damage group than TPCK group and flow cytometry apoptosis provethat apoptosis cells is obviously lower in the TPCK groups. Conclusion: TPCK can reducethe degree of oxidative damage in rat lens.Part III The function of UPP on damaged protein in lens epithelial cells Objective: To investigate effects of overexpressed Ubc4for impaired lens protein inlens epithelial cells. Method: Using cultured rat lens epithelial cells same as the first partto evaluate the ability of cells against environmental oxidative stress after transfectedup-regulation factor siRNA Ubc4. Result: The damaged αA1-162crystallin (is a degradationfragment which is easily precipitated of αA crystallin which is a protective protein in lensthat may reduce the role after suffering damage) level significantly reduced aftertransfected siRNA Ubc4which can up-regulate UPP activity compared withnon-transfected cells. Conclusion: The content of αA crystallin degradation fragment(αA1-162crystallin) after transfected siRNA Ubc4to lens epithelial cells which can preventdamaged lens protein from aggregation and precipitation and then this will play aprotective effect in lens under oxidative damage.