| Glaucoma is the second leading cause of blindness worldwide.It is characterized by optic disk changes and progressive visual field loss, and eventually leads to the irreversible apoptosis of retinal ganglion cells. Pathological elevated intraocular pressure (IOP) is the most important risk factor and currently the only controllable factors in the process of prevention and treatment of glaucoma. High IOP usually occurs as a result of an increase in the aqueous humor outflow resistance of the trabecular meshwork (TM). The TM is resident in the anterior chamber angle which composed of trabecular beams made of extracellular matrix (ECM) elements, including fibronectin, laminin and collagen. Cells that line the trabecular beams are believed to be essential for regulating the aqueous humor outflow that controls IOP. Trabecular meshwork cells have a variety of physiological function include chould secrete, synthesis and degradation. The normal morphological structure of trabecular meshwork cells is crucial for maintain the intraocular pressure. Recently, many studies had certify, TM abnormalities are the most common pathogenesis of glaucoma. Still, the pathogenesis of glaucoma is unclear as is the reason why the TM fails to maintain normal levels of aqueous humor outflow resistance. Oxidative stress and vascular damage are considered two major alterations in the TM related to glaucoma.Oxidative stress refers to a state in which the production of reactive oxygen species (ROS) exceeds an organism’s neutralizing capacity, which disrupts the physical equilibrium and leads to a series of pathological changes. Under physiological conditions, the production and elimination of ROS are in equilibrium; however, some xenobiotics, ionizing radiation, may cause the production of ROS to levels that exceed the neutralizing capacity of an organism, thus leading to oxidative stress. It is a prominent feature of many acute and chronic diseases and is the common pathway of many damage.Chloride ion is one of the most abundant anionic in organisms, It widely expressed in all tissue and cells in the body. ClC (voltage-gated chloride channel) is a members of the chloride channel family which is widely expressed all kinds of tissues and organs in mammas. ClC-2 chloride channel is one of the subtypes of ClCs which is the most depth studied in chloride channel family. It is reported to involved in many physiological activities, but less study involved the effect of ClC-2 on oxidative stress.In our previous studies, we also found ClC-2 Chloride channel involved in the proliferation, migration, apoptosis processe of retinal pigment epithelium cells and RGC-5 cells, relevant with the skeleton structure of TM cells.Mithchondrial apoptotic pathway play an important role in variety of eye diseases. It is TM cells mainly endogenous apoptosis pathway, too. In this pathway, Bcl-2 gene family is essential in activating and regulating of apoptosis.In view of this,we design to use H2O2 induce oxidative damage in TM cells,observe the effect of ClC-2 in the TM cells under H2O2 induced oxidative damage and discuss the possible mechanism.In this study, we use primary human trabecular meshwork cells that are brught from Sciencell. Use H2O2 as a inducer to induce oxidative damage. First we used qRT-PCR (real time quantitative polymerase chain reaction) and western blot to verify the expression of ClC-2 in the primary human TM cells from the mRNA and protein levels, then we confirmed the location of ClC-2 in primary human TM cells by immunofluorescence. Then we use concentration gradient H2O2 stimulate TM cells to find the of suitable concentration to made the oxidative stress model, and finally chose 600μM as the target concentration for the further experiment.Primary TM cells were recovery and cultured to the 3-5 passage, after the cells adherenced for more than 24 hours,600μM H2O2 in FBS(Fetal Bovine Serum)-free TMCM (trabecular mechwork cell medium) was added and treated for 2 h to made the TM cells oxdative stress model. cDNA (complementary DNA) and RNAi (RNA interfere) were used to increase/knock down the expression of ClC-2 gene in TM cells. CCK-8 (cell counting kit-8) was used to observe the TM cell activity in each group. FCM (flow cytometer) was used to test the apoptosis rate and intracellular ROS levels in each group, fluorescence microscope was used to de tect the the change of MMP (mitochondrial membrane potential) in each group. We found 600μM H2O2 treat for 2h could lead to the TM cells activity decrease, apoptisis rate increase, intracellular ROS increase and MMP decrease. After enhancing ClC-2 protein expression, cDNA group showed an increase of cell activitie, a reduce of the apoptosis rate, an increase of intracellular ROS and a increase of mitochondrial membrane potential compared with simple H2O2 group. Whereas the ClC-2 knocked down group showed the opposite results.The above experiments confirmed that the ClC-2 plays a protective role in protecting HTM cells from H2O2 induced oxidative damage, by means that enchance the expression of ClC-2 could protect TM cells from H2O2 induced oxidative damage, whereas inhibit ClC-2 experssion would lead the cells more vulnerability from H2O2 induced oxidative stress.In order to further explore the mechanism of ClC-2 protecting HTM cells from oxidative damage, we compared the Bcl-2, Bax and cleaved caspase-3 protein expression level in each groups.The results showed that H2O2 reatment could lead a reduced of Bcl-2 expression level, an increased of Bax and cleaved caspase 3 expression level which would promote the occurrence of apoptosis.And in cDNA transfection group, the Bcl-2 expression level drop increased, Bax and cleaved caspase 3 expression level decreased compared with H2O2 group. Whereas RNAi group showed the opposite trend.The above experimental results illustrate that ClC-2 could protect TM cells from oxidative damage induced by H2O2.The mechanism of this effect may relevant to the mitochondrial apoptotic pathways,and Bcl-2/Bax participate in the regulation process.In conclusion, we discusses the effect of ClC-2 on H2O2 induced oxidative damage in human TM cells, we found during this process, ClC-2 could protect cell activity, reduce the cell apoptosis rate, reduce the intracellular ROS and maintain the mitochondrial membrane potential.Our results suggest that targeting ClC-2 may provide a novel way to inhibit glaucoma and protect trabecular cells from oxidative damage. The mechanism of action of the ClC-2 may associate with the regulation of Bcl-2/Bax regulated mitochondrial apoptosis pathway... |
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