动物造模,N-乙酰半胱氨酸酰胺 z-bo 2022-02-04 430

　　Background: The lens that affects vision gradually loses transparency and forms cataracts. The pathogenesis of cataract is multi-factorial, and the mechanism involved is not fully understood. Oxidative stress is the primary cause of cataract formation. Oxidative stress means the imbalance between the production rate of oxidants and the detoxification rate, and the production rate is significantly higher than the detoxification rate of antioxidants. The organism uses the natural antioxidant defense system to counteract this situation, but with age, oxidants accumulate, and the antioxidant defense gradually weakens, which may be the most important mechanism for the formation of age-related cataracts. Shearer proposed that oxidative stress is involved in sodium selenite-induced cataracts and leads to cataract formation. It is speculated that the oxidative damage caused by selenite may involve the key sulfhydryl calcium oxide ATPase or ion channels. This may lead to changes in lens epithelial cell metabolism and loss of calcium ATPase activity, so calcium accumulates in the lens nucleus and can activate m-calpain. The activation of M-calpain initiates the hydrolysis of β-crystallin, leading to the dissolution of β and α-crystallin, the co-precipitation of γ-crystallin, and finally the formation of cataracts. Since oxidative stress is related to the formation of cataracts, the application of antioxidants that can successfully penetrate the lens tissue is a logical way to eliminate the occurrence of oxidation-related cataracts. The application of antioxidants in the treatment or prevention of oxidative stress-related diseases is becoming more and more popular, and sulfhydryl antioxidants, such as GSH, cysteine and NAC, have shown certain protective effects. However, the negative charge of NAC molecules at physiological pH makes it impermeable to cell membranes. This leads to the use of higher doses and extended treatment plans. N-acetylcysteine amide (NACA) is an analogue of NAC, which has been shown to be more effective than NAC, due to its neutral amide group, increasing its lipophilicity, and therefore, it penetrates the cell membrane. This has been confirmed in previous studies, and its ability to chelate Cu2+, scavenge free radicals and prevent oxidative stress. Based on these results, the main purpose of this study is to evaluate the ability of NACA to prevent and/or reverse sodium selenite-induced cataract formation in Wistar rats. We prove that NACA injection in the intraperitoneal cavity at 9, 11, and 13 days after delivery helps prevent the formation of dense nuclear cataracts. Continuous administration of NACA (from the 15th day to the 30th day after delivery) as an eye drop formulation can reverse the density of cataracts. Our results show that NACA administration can inhibit the activation of M-calpain by supplementing GSH, inhibit lipid peroxidation and prevent calcium accumulation, and ultimately retain lens lens protein, thereby enhancing the antioxidant capacity of selenite-administered animals. defense. Therefore, NACA is expected to be a candidate for the development of drug-based cataract treatments.

　　Method: Experimental design: 40 2-day-old male juvenile Wistar rats were raised in an environment with a temperature of about 22 degrees and a humidity of about 55%, and a 12/12 light-dark cycle environment. Eat and drink freely, and use them for testing after one week of acclimatization. The rats were divided into four groups, (1) control group, (2) NACA, (3) sodium selenite, and (4) NACA + sodium selenite, so that each group had 10 lactating female rats Male cubs. All rats in the NACA and NACA + sodium selenite groups were intraperitoneally injected with NACA (250 mg/kg) on the 9, 11, and 13 days after birth to study the preventive effect of NACA on sodium selenite-induced cataracts. The control group and sodium selenite group received intraperitoneal injection of phosphate buffer (pH 7.4). On day 10 postpartum, all rats with sodium selenite and NACA + sodium selenite received intraperitoneal injection of sodium selenite (19 μmol/kg), while the control group and NACA group received the same amount of phosphate buffer (pH 7.4). Prepare 1% NACA eye drops with phosphate buffer (pH 7.4). Starting from the 15th day after birth (the day the rat opened its eyes) and continuing to the 30th day after delivery, the reversal effect of NACA on the cataract grading of the NACA+sodium selenite group was examined. Before starting the eye drops (15th day after delivery) and on the last day (30th day after delivery), the cataract grading was checked with a slit lamp microscope. After the last administration of NACA or buffer eye drops, ketamine (80 mg/kg) and xylazine (15 mg/kg) were injected intraperitoneally, and all rats were anesthetized for 2 to 3 hours. The weight of the rat pups was measured at the beginning and end of the study. The lens was taken out immediately after euthanasia and placed on dry ice immediately. The samples were stored at a temperature of 80°C for further analysis.

　　Rat eye morphological observation: The morphological examination of rat eye is similar to the procedure described by Carey et al. One hour before the examination, 2.5% phenylephrine hydrochloride and 1% tropicamide eye drops were instilled to start mydriasis. Carry out 10 times magnification inspection with slit lamp microscope. Take representative photos. Cataracts are graded according to the following proportions: transparent lens, level 0; mild lens opacity, level 1; partial nuclear opacity of the lens, level 2; lens dense nuclear opacity, level 3.

　　Glutathione level determination: According to the HPLC method developed by our laboratory, the GSH level in the lens tissue is determined and described. Simply put, each lens is homogenized in serine borate buffer (pH 7.8), then centrifuged at 4°C for 5000×10 minutes, and the supernatant is taken and diluted. Add 50ul of the diluted supernatant to 200ul of nano-water. Add 750 µl of 1 mM NPM in acetonitrile to each sample. The derivatization reaction was carried out at room temperature for 5 minutes, and then 10 μl of 2 N HCl was added to quench the reaction. Each sample was transferred to an HPLC bottle through a 0.45 μm pore filter. Perform analysis.

　　Glutathione and glutathione disulfide (GSSG) content determination: reversed-phase high performance liquid chromatography was used to determine the total glutathione content, to prepare a lens homogenate, and to analyze GSH according to the above method. Then 50μL of the diluted supernatant was added to 60μl of NaDPH (2 mg/ml) nano water. Add 20μL of glutathione reductase (1 unit/ml) and incubate at room temperature for 5 minutes. After incubation, add 120ul of nano water, and then add 750ul of 1 mM NPM for derivatization. After 5 minutes at room temperature, 10 μl of 2 N HCl was added to quench the reaction. Use reversed-phase high performance liquid chromatography for analysis.

　　Result: NACA and sodium selenite affect the formation of lens cataract: intraperitoneal injection of sodium selenite (19μmol/kg) on the 10th day after delivery is sufficient to induce cataract formation, which can be seen when the rat opens its eyes. Observing the eyes of rat pups with a slit lamp microscope, it was found that all animals injected with sodium selenite had cataracts: 20% formed secondary cataracts, and 80% formed tertiary cataracts. In contrast, intraperitoneal injection of NACA reduced the severity of cataract formation; eight out of ten rats (80%) developed grade 2 cataracts, while 1 rat developed grade 1 cataracts, and 1 rat did not. Any occurrence of cataract (grade 0). These results indicate that NACA is successful in preventing cataract formation. By the 4th week, the grade of cataract in rats injected with sodium selenite did not change. Eight out of ten rats (80%) had grade 3 cataract, and 2 rats had grade 2 cataract. However, NACA eye drops treatment reduced the severity of cataract formation. Among ten cases (50%), five rats had grade 2 cataracts, four rats had grade 1 cataracts, and one rat had no cataracts (0 level).

　　The effect of NACA on the levels of lens GSH and GSSG: The content of GSH in the lens of the sodium selenite group was significantly lower than that of the control and NACA groups. Compared with sodium selenite group, NACA + sodium selenite group, NACA treatment significantly increased GSH level. Compared with the control group, the GSSG content of the lens of the sodium selenite group was significantly higher than that of the control group. However, NACA treatment (NACA+sodium selenite) cannot reduce GSSG levels to the control. The ratio of GSH to GSSG in the lens of the sodium selenite group was significantly lower than that of the control group, while the ratio of GSH/GSSG in the NACA treatment group (NACA+sodium selenite) was close to that of the control group.

　　Conclusion: Data shows that oxidative stress plays a role in the formation of cataracts, especially the maintenance of glutathione. Therefore, GSH prodrugs are effective as therapeutic agents for preventing and reversing cataracts. The data supports our hypothesis that NACA protects by increasing GSH, reduces MDA levels, restores enzyme activity, and normalizes calcium levels. Our future research will focus on studying the effectiveness of NACA eye drops and its pharmacokinetic characteristics in different animal models of cataract formation.