| Diabetes is an important chronic diseases seriously threatening human health and quality of life. Diabetes becomes a serious disease following cardiovascular and cerebrovascular diseases and malignant tumors. Under diabetes, the body is in high glucose environment for long-term, resulting in a large vessel, microvascular damage and endangering the heart, brain, kidney, peripheral nerves, eyes, feet and so on. The major complications of diabetes are the diabetic nephropathy, cardiovascular system disease. In eyes, the major complications are diabetic retinopathy, diabetes related uveitis and diabetic cataract. Diabetic cataract is the result of many factors. At present, the major factors include the change of permeability, lens protein glycosylation, pressure oxidation. Under diabetes, the tissue cells receive high glucose stimulation in long-term due to high glucose environment. Then active substances generated by the cells and tissues increased more than antioxidants and scavenging. Oxidative and anti-oxidative are imbalance. Oxidative stress will appear and can directly cause the change of calcium signaling, apoptosis and proliferation in the cells.Hyperlipidemia is a systemic disease caused by abnormal fat metabolism or transport in the plasma resulting that one or more lipids are higher than the normal value. A lot of epidemiological studies have demonstrated that the hyperlipidemia and cataract are very closely related. The value of low density lipoprotein is the main standard of current diagnosis of hyperlipidemia. Under hyperlipidemia, the body often appears high oxidation state. Oxidized low density lipoprotein is increased in plasma. Oxidized low density lipoprotein is one of the most important risk factors of atherosclerosis in patients with hyperlipidemia. Many studies show that high blood cholesterol can affect the calcium signaling, cell proliferation and apoptosis.Calcium ion plays an important role in cell proliferation and apoptosis. Ca2+ is distributed in the intracellular and extracellular fluid. In cells, Ca2+ is mainly stored in the endoplasmic/sarcoplasmic reticulum. The endoplasmic reticulum/ion pumps and ion channels of sarcoplasmic reticulum regulate the uptake and release, thereby forming a fine Ca2+ concentration control system. In cells, Ca2+ as an important second messenger has important significance for the cells to produce physiological and pathological reaction. Recent research results show that Ca2+ channels are widely distributed in various tissues and cells and have an important role in the regulation of physiological and pathological functions in cells. Orai and STIM are two important proteins mediating store-operated Ca2+ entry.This study is divided into two parts to clarify the effects and mechanism of glucose and oxidized low density lipoprotein on store-operated Ca2+ entry, proliferation and apoptosis. Through this study, we can understand the role and molecular mechanism of high blood glucose and oxidized low density lipoprotein in the lens capsule epithelial cells. The study is helpful to understand the role and mechanism of the high blood glucose and oxidized low density lipoprotein in the development of cataract, provides a theoretical basis for the future treatment, and provides new targets for drug screening and potential new methods for clinical treatment.1. The effect of high glucose on Ca2+ store-operated Ca2+ entry, proliferation and apoptosis in lens capsule epithelial cellsCa2+ store-operated Ca2+ entry is evoked by store Ca2+ depletion which then activates the cell membrane Ca2+ channels mediating extracellular Ca2+ influx. Ca2+ store-operated Ca2+ entry discovered in recent years is an important pathway to control the influx of extracellular Ca2+. Ca2+ signaling mediated by Ca2+ store-operated Ca2+ entry may be an important pathway for the information communication within and outside the cells. Therefore, Ca2+ store-operated Ca2+ entry may have an important role in the regulation of proliferation and apoptosis of the cells. In this study, we cultured the lens capsule epithelial cells(HLEpi C cell line) in vitro with high glucose medium to mimic high blood glucose environment. The effect of high glucose culture on apoptosis and proliferation of HLEpi C cells was examined. The results showed that:(1) the expression of Orai1 in the lens capsule epithelial cells of patients with diabetic cataract was increased significantly;(2) the Ca2+ imaging experiments showed that high glucose cultured for 1, 3, 7 and 14 days, adenosine triphosphate(ATP)- and endoplasmic/sarcoplasmic reticulum Ca2+ pump inhibitor thapsigargin(TG)-evoked Ca2+ store-operated Ca2+ entry was increased significantly;(3) Western blot results showed that the expression level of STIM1 and Orai3 in high glucose cultured HLEpi C cells increased significantly; Orai2 did not change significantly; but Orai1 was decreased significantly;(4) Orai1 and STIM1 si RNA significantly inhibited the expression of Orai1 and STIM1 protein in HLEpi C cells, but also significantly inhibited TG-evoked Ca2+ store-operated Ca2+ entry;(5) After the HLEpi C cells were cultured in high glucose for 7 days, the proliferation and apoptosis were significantly enhanced. These results indicate that the enhanced Ca2+ store-operated Ca2+ entry induced by high glucose culture may be due to the increased expression of the proteins forming Ca2+ store-operated Ca2+ entry in HLEpi C cells. The enhanced Ca2+ store-operated Ca2+ entry induced by high glucose culture might be one important factor to cause the increased proliferation and apoptosis in HLEpi C cells.2. The effect oxLDL on Ca2+ store-operated Ca2+ entry, proliferation and apoptosis in lens capsule epithelial cellsOxidized low density lipoprotein can also affect Ca2+ signaling, cell proliferation and apoptosis. This study used copper sulfate to oxidize low density lipoprotein to prepare oxidized low density lipoprotein. After then according to the literature we used 100 μg/ml oxidized low density lipoprotein to treat HLEpi C cells for 24 h. Ca2+ store-operated Ca2+ entry, the proliferation and apoptosis of HLEpi C cells were examined. We found that:(1) Ca2+ store-operated Ca2+ entry evoked by TG was increased;(2) Western blot results showed that the expression level of Orai3 and STIM1 proteins were significantly increased in oxidized low density lipoprotein treated HLEpi C cells. But the expression level of Orai1 and Orai2 did not have significant change;(3) CCK8 cell proliferation results showed that the proliferation in oxidized low density lipoprotein treated cell was enhanced significantly;(4) the TUNEL experimental results also showed that the apoptosis of HLEpi C cells treated by oxidized low density lipoprotein was significantly enhanced. These results suggest that oxidized low density lipoprotein can lead to increased expression of the proteins forming Ca2+ store-operated Ca2+ entry, which then caused enhanced Ca2+ store-operated Ca2+ entry. In addition, the enhanced Ca2+ store-operated Ca2+ entry may be one the reasons that cause the increased cell proliferation and apoptosis of HLEpi C cells. |
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