The Protective Effect And Mechanism Of Berberine On Ovalbumin-induced Allergic Conjunctivitis

Objections:In this study,we investigated the protective effect of berberine（BBR）on Ovalbumin（OVA）induced Allergic conjunctivitis（AC）in mice,and the mechanism of its action was also preliminarily studied.Methods:1.methods of comparison for preparing mice models of allergic conjunctivitis with different OVA challenge concentrationsFemale BALB/C mice were selected,and antigen adjuvant suspension（containing OVA 50μg,AL（OH）₃2mg）prepared by intraperitoneal injection of normal saline into the mice on the 0th,7th and 14th days of the experiment 200μL for systemic sensitization.From the 15th day to the 24th day of the experiment,5% or 10% OVA solution was dripped into both eyes every day for challenge.After recording the number of eye scratches on the 24th day of the experiment,the ocular symptoms were scored,and the mice were sacrificed 24 hours after the last challenge.The serum levels of OVA-specific IgE（OVA-s IgE）and histamine（Histamine,HIS）were determined.After recording the number of eye scratches on the 24th day of the experiment,the ocular symptoms were scored,and the mice were sacrificed 24 hours after the last challenge.The serum levels of OVA-specific IgE（OVA-s IgE）and histamine（Histamine,HIS）were determined.2.Methods for the protective effect of berberine on OVA induced allergic conjunctivitis in miceThe model of allergic conjunctivitis was established under the condition of optimal OVA concentration.10μL of berberine（0.125,0.25,and 0.5 mg/m L）solution was administered to each of the eyes of both sides 1 hour before OVA challenge every day,and the number of eye scratches in the mouse 10 min after OVA challenge was recorded.Recorded on the 24th day of the experiment,the number of eye scratches of the mice were,the ocular symptoms were counted.The mice were sacrificed 24 hours after the last OVA challenge,and the contents of OVA-s IgE,HIS,Interleukin-4（Interleukin-4,IL-4）and Interleukin-13（Interleukin-13,IL-13）in the mouse serum were determined.The mouse conjunctival tissues were stained with HE and toluidine blue,and the pathological changes,the distribution of mast cells and the changes of degranulation of the conjunctival tissues of the mice were observed under the microscope.Furthermore,Western Blot was used to determine the expression of MAPK pathway proteins ERK,JNK and p38 in the conjunctival tissue of mice with allergic conjunctivitis.Result:1.Compare the results of using different OVA concentrations to challenge the preparation of mice models of allergic conjunctivitis（1）Compared with the normal control group,the number of eye scratching in the5% OVA model group and the 10% OVA model group increased significantly on day4-10 th（P<0.001）.Compared with the 5% OVA model group,the number of eye scratches in the 10% OVA model group increased significantly on day 4-10 th（p<0.001）.（2）Compared with the normal control group,the eye symptom scores of the 10% OVA model group and the 5% OVA model group were significantly increased（P<0.001）.Compared with the 5% OVA model group,the eye symptom score of the mice in the 10% OVA model group was significantly increased（p<0.001）.（3）Compared with the normal control group,the serum histamine and OVA-specific IgE levels in the 5% OVA model group and the 10% OVA model group increased significantly（p<0.05）.2.Effect of berberine in allergic conjunctivitis mice（1）Effect of berberine on ther eye scratches numbe in miceAfter OVA challenge,the number of eye scratches in the model group was significantly higher than that in the normal control group（p<0.01）.Compared with the model group control,the number of eye scratches in the berberine administration group decreased（p<0.05）.（2）Effect of berberine on the ocular symptoms in allergic conjunctivitis miceCompared with the normal control group,the model control group mice have higher symptom scores.Compared with the model control group,high and medium doses of berberine can significantly reduce the ocular symptom scores of mice（p<0.01）,and low doses of berberine can reduce the ocular symptom scores of mice（p<0.05）.（3）Effect of berberine on the serum OVA-specific IgE,HIS,IL-4 and IL-13levels in allergic conjunctivitis miceCompared with the normal control group,the OVA-s IgE and HIS levels of the model control group mice were significantly increased（p<0.01）.Compared with the model control group,the serum OVA-s IgE,HIS,IL-4 and IL-13 levels of mice in the high-dose and medium-dose groups of berberine were significantly reduced（p<0.001）.The mouse serum OVA-s IgE level was significantly reduced（p<0.01）,and the levels of HIS,IL-4 and IL-13 were reduced（p<0.05）.（4）Effect of berberine on HE staining of conjunctival tissue in allergic conjunctivitis miceHE staining results showed that the conjunctival tissue structure of the normal control group was clear and complete.There was no inflammatory cell infiltration in the lamina propria and subcutaneous tissue of the conjunctiva,and mast cells were occasionally seen.In the model control group,severe conjunctival tissue edema and conjunctival hyperemia were seen.The number of eosinophils in the lamina propria and subcutaneous tissue of the conjunctiva increases,and a large number of small blood vessels proliferate under the skin.Compared with the model control group,each administration group of berberine has a improvement effect.（5）Effect on toluidine blue staining of conjunctival tissue in allergic conjunctivitis miceThe results of toluidine blue staining showed that the number of mast cells in the conjunctival tissue of the model group mice increased,and the degranulation rate of mast cells increased.Compared with the model control mice,the number of conjunctival mast cells in the high-dose and medium-dose groups of berberine was significantly reduced,and the degranulation rate was significantly reduced（p<0.001）.The number of conjunctival mast cells in the low-dose berberine group decreased,and the degranulation rate decreased（p<0.05）（6）Effect of of MAPK signaling pathway-related proteins of conjunctival tissue in OVA induced allergic conjunctivitis miceCompared with the normal control group,the protein expression of the MAPK pathway phosphorylated proteins p-ERK,p-JNK and p-p38 in the conjunctival tissue of the model control group increased（p<0.05）.Compared with the model control group,the expression levels of p-ERK,p-JNK and p-p38 in the berberine group decreased（p<0.05）.Conclusion:1.Berberine can improve the symptoms of allergic conjunctivitis,reduce the levels of OVA-s IgE,HIS,IL-4 and IL-13 in the serum,relieve the edema,congestion and inflammatory infiltration of conjunctival tissue,and inhibit conjunctival mast cells Activation and degranulation.2.The effect of berberine in the treatment of allergic conjunctivitis may be through Inhibit the phosphorylation level of MAPK pathway related proteins ERK,JNK and p38.