Research On The Intervention And Mechaninsm Of Shuangqin Eye Gel On Herpes Simplex Keratitis (HSK) In Experimental Mice

Objective1. To explore the intervention of Shuangqin eye gel on herpes simplex keratitis (HSK) in experimental mice.2. To evaluate the anti-relapse effect of Shuangqin eye gel intervention in experimental mice with herpes simplex keratitis (HSK)3. To explore the intervention of Shuangqin eye gel on recurrent herpes simplex keratitis (HSK) in experimental mice.Methods1. The corneas of BALB/c mice were inoculated with herpes simplex virus type 1 (HSV-1) SM44 strain. The mice were randomly divided into three groups: controls, mice treated with acyclovir (ACV) and Shuangqin eye gel respectively, all of whom were administered continuously for two weeks. The efficacy and mechanism can be evaluated through scoring the lesion symptoms, observing the corneal tissue pathological slices and detecting the expression of the LAT in the TG with reverse transcription polymerase chain reaction (RT-PCR)2. The corneas of BALB/c mice were inoculated with herpes simplex virus type 1 (HSV-1) SM44 strain. The mice were randomly divided into three groups: controls, mice treated with acyclovir (ACV) and Shuangqin eye gel respectively, all of whom were administered continuously for two weeks.8 weeks after stopping treatment, the corneas were irradiated by ultraviolet B (UV-B) light in order to induce the relapse of HSK. Then,we evaluate the effect by the following methods:observing the relapse rate of each group,scoring the lesion symptom after irradiating by UV-B light and detecting the hiding of HSV-1 and the expression of the latency-associated transcript (LAT) in the trigeminal ganglion (TG) by RT-PCR.3. The corneas of BALB/c mice were inoculated with herpes simplex virus type 1 (HSV-1) SM44 strain. The mice were randomly divided into three groups:The gel matrix group, acyclovir (ACV)group、Shuangqin eye gel group and cortisone group.After 6 weeks, the corneas were irradiated by ultraviolet B (UV-B) light in order to induce the relapse of HSK.and all of whom were administered continuously for two weeks.Then,we evaluate the effect by the following methods:observing and scoring the corneal and conjunctival symptom after irradiating by UV-B lightl,3,5,7, 10,12,14 days;observing the corneal structural changes by HE stain;observing the expression of the nterleukin-17 (IL-17) antibody positive cells by immunohistochemical method;detecting the contents of tumor necrosis factor-a (TNF-a)in the cornea.Results1. After the treatment 14 days, there were less scores on the lesion symptoms in Shuangqin eye gel group and ACV group than the control group, and the difference was statistically significant (p<0.01); Meanwhile the difference between Shuangqin eye gel group and ACV comparison group was also statistically significant (p<0.05); Through observing the corneal tissue pathological slices, the corneal layers structures of Shuangqin eye gel were generally normal and the difference among Shuangqin eye gel group, the control group and ACV group was statistically significant (p<0.05); Through detecting the semi-quantitative RT-PCR gray value of the LAT mRNA in TG in each group of the mice, the results show that the inhibition of Shuangqin eye gel to the expression of the LAT in TG is superior to the control group (p<0.05), which in ACV group is not obvious.2. All virus detection rate of tear film cleaning fluid of HSK mice in the three groups were 0.00% in the first day, and subsequently the detection rate of the three groups were increased with different degrees. In 7 days after irradiating by UV-B light, the virus detection rates of tear film cleaning fluid of HSK mice, the control group were the highest. the virus detection rates of Shuangqin eye gel had been consistently lower than the other two groups expect Shuangqin eye gel group higher than ACV group. But comparing the virus detection rates of tear film cleaning fluid of HSK mice, the difference was not statistically significant;Through observing the score of corneal lesions, the corneal lesions of Shuangqin eye gel group after irradiation by UV-B light were lighter than the other two groups (p<0.05),and the difference was statistically significant; Through observing the corneal tissue pathological slices after irradiation by UV-B light, the corneas of the control group and the ACV group were damaged with varying degrees while the corneal layers structures of Shuangqin eye gel were generally normal; Through detecting the PCR gray value of the HSV-1 DNA in TG in each group of the mice, the results show that Shuangqin eye gel group is superior to the control group(p<0.05) and the ACV group (p<0.01); Comparing the RT-PCR gray value of the LAT mRNA in TG in each group of the mice, the results also show that Shuangqin eye gel is superior to the control group (p<0.01) and the ACV group (p<0.05).3. According to the results of the corneal lesions in mice in the different time points after UV light-B change, the corneal disease score of groups are still in the declining trend; after irradiation 3 days, comparing the gel matrix group and Shuangqin eye gel group, the difference was statistically significant; after irradiation 12 and 14 days, the cornea pathological of Shuangqin eye gel group、ACV group and cortisone group were dropped sharply, and compared to gel matrix group, the differences were statistically significant; according to the results of the treatment of conjunctival lesions in mice after UV light-B 2 days change, compared to gel matrix group, the differences of Shuangqin eye gel group、the ACV group and cortisone group were statistically significant;Through observing the corneal tissue pathological slices by HE stain,the corneal tissue of Shuangqin eye gel group show that the substrate layer were Mild edema, but also their structure roughly normal and the corneal protective effect in mice of Shuangqin eye gel group was obvious than the rest of the several groups; through the expression of the IL-17 by immunohistochemical method,compared to gel matrix group, the differences of Shuangqin eye gel group and ACV group were statistically significant;through the contents of TNF-α in the cornea,compared to gel matrix group, the differences of Shuangqin eye gel group and the gel matrix group were statistically significant; Immunohistochemical method to detect the cornea of recurrence of each type of HSK mice IL-17 resistance.Conclusion1. Shuangqin eye gel can not only alleviate the symptoms of HSK and protect the cornea of normal tissue structure, but also inhibit the expression of LAT in the TG.2. The virus detection rate of HSV-1 can be reduced by Shuangqin eye gel to a certain extent, alleviate the symptoms of HSK after UV-B irradiation, protect the normal tissue structure of corneas, but also inhibit the latency of HSV-1 and the expression of LAT in the TG.. Meanwhile, but its anti-.relapse and mechanism have yet to be further explored.3. Shuangqin eye gel has a certain therapeutic effect for recurrent herpes simplex keratitis and play the role of protecting corneal and conjunctival、reducing damage and maintaining the integrity of the structure of the cornea to a certain extent in the early days of the lesions.we suspect that Shuangqin eye gel play the roles by inhibiting the cornea in the expression of IL-17, reducing the content of TNF-α in the cornea and reducing the cell apoptosis by the inhibition of TNF-α.