| ObjectiveUnder the condition of the oxidative stress induced by hydrogen peroxide,observe the different concentrations of Bu Qing granule in culture in vitro of rat lens the intervention of the role and influence on protein TTase?thioltransferase?and Trx?thioredoxin?.In order to provide experimental basis for Bu Qing granule to prevention and treatment of age-related cataract?ARC?.Methods1)Preparation of drug-containing serum:40 SPF 8-week-old male SD rats were randomly divided into 5 groups:blank serum group?1 mL/100 g physiological saline?and positive control group(1 g·m L-1 Qiju Dihuang Wan aqueous solution),high-,medium-,and low-concentration BuQing granules(4 g·mL-1,2 g·mL-1,and 1 g·m L-1 respectively)of aqueous solution,8 in each group,twice daily Intragastric administration,continuous administration for 4 days,blood collection,centrifugation,and the supernatant were taken as drug-containing serum.2)60 healthy SD rats,120 eyes,male.After anaesthesia with 7%chloral hydrate?0.6m L/100g?in the abdominal cavity,the rats were sacrificed and the lens was removed.The lens was placed in a carbon dioxide incubator at 37°C and 5%CO2 for 8 hours and then the cloudy lens was discarded.The remaining lenses were randomly divided into 6 groups of 16in each group:blank control group,model group,Qijudihuang Wan group,high concentration group of BuQing granule,medium concentration group of BuQing granule s,and low concentration group of BuQing granule.3)Observe the turbidity of each lens group.HE staining was used to observe the pathological changes of each lens group.The thermal stability of each lens protein was measured.The TTase and Trx levels in each lens group were measured by ELISA.Real-time Quantitative Polymerase Amplification Reaction?Real Time-PCR?was used to detect the expression of TTase and Trx in RNA at the RNA level,and western blot was used to detrmine the protein expression of TTase and Trx in the lens.Results1)Lens turbidity:The lens of the blank group remained transparent during the entire incubation process and could completely penetrate the“+”line.In the other groups,the lens began to show different degrees of cortical opacification with the prolongation of incubation time.In the model group,the“+”line was completely absent,and the lens was completely turbid.The remaining lenses in each drug-containing serum group were clearer than those in the model group.The lens in the BuQing granule high-concentration group were clearer and could pass through the“+”line.2)HE staining:In the blank group,the cell morphology was regular and the fibers were tightly arranged.The lens epithelial cells of the model group had irregular morphology,different shades of staining,disordered arrangement,and ruptured cytoplasm and more vacuoles.The rest of the drug-containing serum groups were less than the lens tissue damage in the model group.Among them,the lens epithelial cells in the BuQing granule high-concentration group were arranged more neatly and the cytoplasm was relatively intact.3)Thermal stability of protein:The OD value of the blank group was always stable,and the OD values of the other groups were all increased.Excluding the blank group,the OD values in each group were slowly increased from 50°C to 56°C,and the trend was relatively stable.There was no significant difference between the groups.In the 62°C to 86°C range,the OD value increased rapidly,the trend was steeper,and the lens protein was mixed.The turbidity of the supernatant was obvious and the turbidity was faster.The difference between the model group and the blank group was significant?P<0.01?.Compared with the model group,the OD value of each group was more stable and there was a significant difference?P<0.05 or 0.01?.4)Compared with the blank group,the activity of TTase and Trx model group was significantly lower than that of the blank group?P<0.01?.Compared with the model group,the activity of the high concentration of Buqing Granule group was significantly higher than that of the model group.The difference was statistically significant?P<0.01?.Compared with the model group,the concentration of Buqing granule in the medium concentration group and the the low concentration group was higher than that in the model group?P<0.05?.5)Western blot:The results showed that compared with the blank group rats,the expression level of the lens in the model group was significantly decreased?P<0.01?;compared with the model group,the protein expression was observed after the administration of the Buqing Granules drug-containing serum.The level increased?P<0.01?.Trx:Compared with the blank group,the expression level of the lens in the model group was significantly decreased?P<0.01?.Compared with the model group,the expression level of the Buqing Granules drug-containing serum was increased?P<0.01?.6)Real-Time PCR:The results of real-time fluorescence quantitative PCR showed that the TTase mRNA level in the lens of the model group was significantly lower than that of the blank group?P<0.01?.Compared with the model group,after intervention with Buqing Granule drug-containing serum,the TTase mRNA level in each group increased significantly?P<0.01?.Compared with the blank group,the level of Trx mRNA in the lens of the model group was significantly decreased?P<0.01?.Compared with the model group,after intervention with Buqing Granule drug-containing serum,the Trx mRNA level in each group was significantly higher than that of the model group?P<0.01?.Conclusion1.Oxidative stress may be the one of the main mechanisms of cataract.2.Buqing Granules can delay the turbidity of rat lens caused by oxidative stress,and have a good protective effect on the tissue structure.3.The H2O2 may up-regulate the expression of TTase and Trx protein in the lens,and prevent and delay the occurrence and development of lens opacification induced by H2O2 in vitro.4.Bu Qing granule with high concentration of 4g·m L-1 and complementing granules with concentration of 2g·m L-1supplemental granules in treating ocular opacity caused by oxidative stress is better.4.The effect of high concentration of 4g·m L-1 and medium concentration of 2g·mL-1 in Bu Qing Granule is better than that of turbidity induced by oxidative stress in rats. |
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