动物实验,角膜炎症 z-bo 2020-09-08 421

　　Introduction: Keratoconjunctivitis sicca is a pathological disease, usually called dry eye, which is characterized by changes in the amount and quality of tear secretion. The clinical manifestations are infection, irritation, ulcers, and blurred vision. The ocular surface integrity of patients with dry eye is affected by many factors such as age, airflow, ultraviolet light, temperature, chemical composition, humidity, hormones and so on. All these changes generate free radicals that induce inflammation and oxidative stress. The reported literature confirms that tear fluid contains a variety of antioxidants, such as lactoferrin, tyrosine and glutathione, which protect corneal epithelial cells. Lack of tears on the ocular surface and dehydration lead to dry eye syndrome. Therefore, the inflammation and oxidative stress of corneal ulcers caused by dry eye are increased. Therefore, drugs that inhibit these factors may be a treatment option for dry eye. Estrogen and its derivatives have strong antioxidant and anti-inflammatory effects. The study by Hiji et al. proved that 2-hydroxyestradiol can significantly reduce inflammation and oxidative stress in a rat model of dry eye. It is known that isoflavones have strong antioxidant and anti-inflammatory activities, and isoflavones such as daidzein and genistein have similar effects to estrogen. In recent years, many medicinal plants and their metabolites have been used as substitutes for the treatment of various antibiotics and therapeutic agents. The literature shows that daidzein is used for the treatment of alcohol dependence and neurodegenerative diseases based on its antioxidant and anti-inflammatory effects. Daidzein inhibits inflammatory mediators by inhibiting the activation of NF-κB and STAT-1. This study investigated the role of daidzein in the treatment of dry eye.

　　Animals: Healthy male Wistar rats (250～300 g) were used for pharmacological screening in this study at 8 weeks of age. Evaluation of the free radical scavenging activity of daidzein: The tyrosine free radical was estimated according to the previously reported electron spin resonance study. The mixture PBS (pH 7.4) used in this study contains 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, 100 mM), H2O2 and myoglobin (400μM) in ethanol. The sterol is poured into the reaction mixture given above, and then H2O2 is added to start the reaction. ESR spectroscopy was performed at room temperature.

　　"The influence of daidzein on PGS activity: The human corneal epithelial cell line CEPI-17-CL4 was used to evaluate the influence of daidzein on cell viability to further estimate the concentration of daidzein. The HCG supplemented medium is used to cultivate CEPI cells in 96-well plates. The CEPI cells and daidzein were incubated in the medium for one day. Replace the original medium with fresh medium containing 10% Alama Blue. This reagent is used to estimate cell viability. After incubating for one hour, determine. The evaluation of PGS inhibitory activity confirmed the anti-inflammatory activity of catechol drugs. The PGS inhibitory activity of daidzein was evaluated with CEPI cell extract. Homogenize the CEPI cells with Tris buffer (5mM, pH 7.4) containing DTT and EDTA at 10000 rpm for 5 minutes. The PGS activity was measured with COX fluorescence activity assay kit. The activity of COX1 and COX2 enzymes was measured by fluorescence method.

　　The effect of daidzein on dry eye: A rat model of dry eye was used to evaluate the effect of daidzein. The rats were anesthetized and the lacrimal glands were taken to make a rat model of dry eye. The experiment was started on the first day after removal of the lacrimal gland, and the concentration of daidzein was 0.1, 1, 10 μM. Daidzein was prepared at a concentration of 10 mM in PBS of 10, 0.1, 1, and 10 μM. Each rat was given 5 μl of daidzein a day for 2 weeks, four times a day. The rats in the control group were given excipients with lacrimal gland removed. After the treatment, the tear volume and fluorescein score were evaluated. In addition, the ocular surface was stained with fluorescein to evaluate the degree of dry eyes. The corneal photo is divided into nine different areas, and each area scores between 0 and 3 according to the degree of staining. The scores of each area are added together to get the total score, which is called the fluorescein score. Factors such as airflow, temperature and humidity are varied. Take the normal rat score as a reference. Total RNA was isolated from the cornea of the entire group for real-time PCR. TrIZOL was used to extract total RNA from the cornea and perform reverse transcription. RT-PCR was used to detect the expression of heme oxygenase, TNFα, interleukin 6 (IL-6), matrix metalloproteinase 9 (MMP-9) and PGS-2 genes. The COX fluorescence activity assay kit was used to measure the PGS activity in the corneal extracts of the normal group and the control group.

　　"Result: Research on the free radical scavenging activity of daidzein: ESR studies show that the ESR signal of tyrosyl free radicals is reduced by 1 mM ester diol and 1 mM daidzein. In addition, 0.3～0.5mM daidzein also significantly reduced the signal.

　　The effect of daidzein on the activity of PGS: At 10uM, the cell survival rate is as high as 90%, after which the concentration further increases, and the cell viability decreases significantly. 10uM, daidzein has a significant inhibitory effect on PGS activity. However, the other steroids used in this study did not change PGS activity. Therefore, based on its free radical scavenging activity and PGS inhibiting activity, daidzein is used to treat dry eye.

　　The effect of daidzein on dry eye: Fluorescein stained cornea photos show that the area of fluorescein stained with PBS is larger than that with daidzein. The fluorescein score was also observed in this study. The fluorescein score of rats in the treatment group was significantly higher than that in the normal control group. Compared with the vehicle control group, daidzein treatment significantly reduced the score. The results of tear secretion showed that the tear secretion of the treatment group was reduced by about 45% after the lacrimal gland was removed. After the tear gland secretion was reduced, the tear secretion was reduced. In rats without lacrimal glands, the secretion of tears was reduced and the cornea fell off. Compared with the normal control group, the fluorescein score of dry eye patients was significantly increased. The results showed that the PGS activity of dry eyes was higher than that of the normal side. In addition, daidzein significantly inhibits PGS activity compared with dry eye.

　　Discussion: This study evaluated the protective effect of daidzein on the cornea of a rat model of dry eye syndrome, and initially explored the mechanism of daidzein. A rat model of dry eye syndrome was established by removing the lacrimal gland. Various reports indicate that this model is simple for the induction of dry eye, and there is no chemically induced dry eye. The literature shows that in dry eyes, the expression of IL-6, MMP9 and PGS2 in corneal cells increases. Daidzein inhibits the expression of IL6 and MMP9, and the expression of Id6 and MMP9 increases. These results indicate that daidzein improves dry eye syndrome. Daidzein does not affect the expression of PGS2 in CEPI cells. This indicated that daidzein was separated from PGS in the cell extract. Therefore, this study believes that daidzein inhibits the activity of PGS through competition. Its effect on tyrosine free radicals was also evaluated during the investigation. It is observed that daidzein has tyrosine free radical scavenging activity, and tyrosine free radical plays an important role in the activity of PGS. Therefore, there is a certain relationship between tyrosine free radical scavengers and PGS inhibitors. This means that daidzein inhibits PGS activity based on its tyrosine free radical scavenging activity. Daidzein can partially restore tear secretion in dry eye rats. These effects may be due to increased secretion of lacrimal glands in the orbit or restoration of ocular surface retention.

　　Conclusion: The conclusion of this study is that daidzein protects the cornea of a rat model of dry eye by inhibiting inflammation and oxidative stress.