| ObjectiveTo investigate the protective effects and mechanisms of isopsoralen (ISR), a Chinese kidney-tonifying herbal monomer inducing estrogen-like activities, and ecdysterone (ECR) on the human lens epithelial cell (HLEC) with oxidative damage induced by H2O2. Furthermore, to pursue the mitochondrial proteomics mechanism of the protective effects of ISR and ECR on HLEC with oxidative damage. In order to provide scientific basis for pursueing safe and effective natural drugs to prevent and cure senile cataract.MethodsThe oxidative damage model of HLEC duplicated by H2O2, and then ISR, ECR, and HLEC were incubaed with E2as a positive control for the following studies:1. The gene expression of SODmRNA, GSH-pxmRNA and CATmRNA were assayed by reverse transcription-polymerase chain reaction(RT-PCR) to probe the enzymatic mechanisms of ISR and ECR, which protect HLEC from oxidative damage.2. The expression of NF-κB in HLEC were analyzed by flow cytometer (FCM), to discuss the cellular signal transduction mechanisms of ISR and ECR, which protect HLEC from oxidative damage.3. The mitochondria morphology of was observed under transmission electron microscope to investigate the morphological changes in the structure of the sub-cellular level of ISR and ECR, which protect HLEC from oxidative damage.4. The different expressions of mitochondria proteome in HLEC was assayed and analyzed by proteome array and surface-enhanced laser desorption ionization time of flight mass spectrometry(SELDI-TOF-MS) technology to pursue the mitochondrial proteomics mechanism of the protective effects of ISR and ECR on HLEC with oxidative damage.Results1. The effects of ISR and ECR on the gene expression of antioxidant enzyme in HLEC with oxidative damage induced by H2O2are as follows: 1.) SOD mRNA expression:The SOD mRNA expression in HLEC of the H2O2group decreased, with the differential expression from the control group having statistical significance (P<0.01); and, the SOD mRNA expressions of the E2, ISR, and ECR groups are significantly higher than those of the H2O2group (P<0.01).2.) GSH-px mRNA expression:The GSH-px mRNA expression in HLEC of the H2O2group decreased, with the differential expression from the control group having statistical significance (P<0.01); and, the GSH-px mRNA expressions of the E2, ISR, and ECR groups are significantly higher than those of the H2O2group(P<0.01).3.) CAT mRNA expression:CAT mRNA expression in HLEC of the H2O2group decreased, with the differential expression from the control group having statistical significance (P<0.01); and, the CAT mRNA expressions of the E2, ISR, and ECR groups are significantly higher than those of the H2O2group(P<0.01).2, The effects of ISR and ECR on the expression of nuclear factor-κB in HLEC with oxidative damage induced by H2O2are as follows:Normal HLEC has the default NF-κB expression; the NF-κB expression in HLEC of the H2O2group significantly increased, with the differential expression from the control group having statistical significance (P<0.01); the NF-κB expression in HLEC of the NF-κB inhibitor PDTC group significantly decreased, with the differential expression from the H2O2group having statistical significance (P<0.01); and, the NF-κB expressions of the E2, ISR, and ECR groups are between the control group and the H2O2group, and lower than the PDTC group, with the differential expression from the H2O2group having statistical significance (P<0.01).3, The effects of ISR and ECR on the mitochondrial ultrastructures in HLEC with oxidative damage induced by H2O2:The typical morphological changes of damaged HLEC were observed under transmissionelectron microscope, such as Double-layer structure of mitochondrial membrane integrity, ridge structure disappeared, vacuolization of mitochondria; as well in HLEC mitochondria induced by H2O2. However, ISR、ECR and E2group can alleviated these changes of morphology.4, The effects of ISR and ECR on the mitochondrial proteome in HLEC with oxidative damage induced by H2O2.1.) There were fifty protein spots identified on the CM10chip containing the H2O2sample. In two protein spots, with mass/charge (m/z) ratios of6532and6809, expression was up-regulated. The values of those two spots were13.6and4.8times higher, respectively, than the values of the same spots in the control sample. There were significant differences between the mitochondrial proteomes of H2O2exposed cells and the control cells (P<0.05).2.) There were forty-six protein spots on the CM10chip bound by HLEC exposed to E2and H2O2. Expression of the spot with an m/z ratio of6532was down-regulated2.8fold (P<0.05) compared to H2O2alone.3.) There were forty-nine protein spots on the CM10chip bound to samples from HLEC exposed to ISR and H2O2. Expression of the spots with an m/z ratio of6532and6809were down-regulated2.2and1.9fold (P<0.05), respectively, compared to H2O2alone. There were forty-nine protein spots on the CM10chip bound to samples from HLEC exposed to ECR and H2O2. Expression of the spots with an m/z ratio of6532and6809were down-regulated3.4and2.6fold (P<0.05), respectively, compared to H2O2alone.Conclusion1. Chinese kidney-tonifying herbal monomer ISR, ECR, and E2have the same activities and estrogen-like effects and they have protective effects on HLEC with oxidative damage induced by H2O2. The up-regulation of the expressions of SOD mRNA, GSH-px mRNA and CAT mRNA and the down-regulated NF-κB p65may be the mechanisms of ISR and ECR preventing and treating oxidative damage of HLEC induced by H2O2.2. ISR and ECR have protective of mitochondrial ultrastructures in HLEC with oxidative damage induced by H2O2.3. The protein spot with the m/z ratio of6532may be the target for ISR and ECR in protecting HLEC from oxidative damage induced by H2O2.4. Ribosomal protein S29may be an important mitochondrial proteins in the ISR and the ECR to protect HLEC with oxidative damage.5. ISR and ECR have phytoestrogen-like effects on HLEC and cataract. |
PDF Full Text Request