| Allergic conjunctivitis(AC)is one of the most common clinical allergic conditions and is a hypersensitivity reaction caused by frequent exposure of the conjunctiva to allergens in the surrounding environment(pollen,dust,animal hair,etc.).Subjective clinical symptoms include itchy eyes,foreign body sensation,and eye discharge.Epidemiological statistics show that AC affects up to 15-40%of the population each year,with children accounting for 30%.Due to its high incidence,high recurrence rate,long duration of onset,and ineffective treatment,it is one of the most difficult diseases to treat in ophthalmology,causing significant physical and emotional stress to patients,and in severe cases,it is a chronic disease that can last for many years.Conventional clinical treatments include antihistamines,mast cell stabilizers,immunosuppressants,glucocorticoids,and nonsteroidal anti-inflammatory drugs,but the above treatments are sometimes difficult to achieve satisfactory results,have short maintenance periods,are prone to side effects such as hormonal glaucoma,and lack effective drugs to control relapse.There is also a lack of effective drugs to inhibit recurrence.Therefore,prolonging the duration of drug action and effectively preventing recurrence of AC in the long term are important directions in AC research.In recent years,the important role of the MAPK signaling pathway in the pathogenesis of several allergic diseases is becoming clearer.The classic pathogenesis of AC,one of the most common allergic diseases in the world,is thought to be mainly due to a Th1/Th2 immune imbalance following the onset of metaplasia,in which the combination of allergens and IgE antibodies stimulates the proliferation and activation of eosinophils and neutrophils,causing the following symptoms The combination of allergen and IgE antibody The combination of allergen and IgE antibodies stimulates proliferation and activation of eosinophils and neutrophils,leading to mast cell degranulation and increased release of inflammatory mediators(histamine,prostaglandins,leukotrienes,platelet activating factor,etc.),resulting in inflammatory symptoms such as conjunctival edema and eye redness.The Septiherb Alleviating Allergic Conjunctivitis is based on the Chuan Jiao Formula and is optimized with the addition of Serpentine and other ingredients,aiming to avoid drug toxicity so that it can be used safely in clinical practice for a long time.Based on the complex pathogenesis of "cold and heat are combined",the formula was repeatedly refined,cut and formulated.Therefore,the MAPK signaling pathway was selected as the target pathway of The Septiherb Alleviating Allergic Conjunctivitis in this study,in order to reveal the anti-inflammatory immune mechanism of The Septiherb Alleviating Allergic Conjunctivitis,and to provide a theoretical basis for the long-term and safe application of The Septiherb Alleviating Allergic Conjunctivitis in clinical practice.This study was conducted to reveal the anti-inflammatory immune mechanism of The Septiherb Alleviating Allergic Conjunctivitis and to provide a theoretical basis for its wide and long-term safe application in clinical practice.ObjectiveTo investigate the anti-inflammatory immune mechanism of The Septiherb Alleviating Allergic Conjunctivitis on ovalbumin-induced allergic conjunctivitis mouse model based on MAPK signaling pathway.MethodsBALB/c mice were sensitized by intraperitoneal injection of OVA and adjuvant AI(OH)3 mixture on days 0、7 and 14 respectively,and then the eyes were spotted with OVA and saline mixture once a day for 17 consecutive days,and the model was established on the third day of stimulation according to the ocular symptoms.The successfully modeled mice were randomly divided into the model group,The Septiherb Alleviating Allergic Conjunctivitis treatment group(high,medium and low dose groups of Chinese medicine),the positive drug control group(Olopatadine group),the negative control group,and the blank group in accordance with the principle of random grouping.Intervention method:The positive drug control group applied 0.1%olopatadine hydrochloride eye drops,and the Chinese herbal medicine treatment group applied The Septiherb Alleviating Allergic Conjunctivitis intervention treatment for two weeks.Detection method:After the last OVA excitation for 20 min,the eye symptoms were scored,the mice’ heads were fixed and the mice’ eyes were photographed.Blood was collected 24h after the last excitation by eyeball removal method.Serum OVA-sIgE,TNF-α,HIS,IL-4 and IFN-γ were measured by enzyme-linked immunosorbent assay(ELISA).The conjunctiva and eyelid tissues of mice were taken,and the conjunctival eyelid sections of each group were stained by HE and toluidine blue staining,and the pathological changes of the conjunctiva of mice were observed by light microscopy.The expression contents of MAPK signaling pathway-related proteins ERK,P-ERK,JNK,P-JNK,P38 and P-P38 in the conjunctival tissues of mice were determined by Western Blot method.Results1.Histopathological changes of eyelids and conjunctiva and ocular symptom scores were observed under slit lamp:20 min after the last excitation,observation under slit lamp could reveal that the blank group had no allergic symptoms,natural eye opening,normal eyelid thickness,no tearing and discharge,and no photophobia,while the model group and the negative control group showed obvious allergic symptoms than the blank group mice,including eyelid swelling,straining to open the eye,tearing and mucous discharge The positive drug control group and each dose group of The Septiherb Alleviating Allergic Conjunctivitis reduced the eye symptoms to different degrees,among which the low dose group of The Septiherb Alleviating Allergic Conjunctivitis still showed obvious eye swelling,eyelid congestion,a small amount of tearing,and slight difficulty in eye opening;the middle dose group,high dose group of The Septiherb Alleviating Allergic Conjunctivitis and positive drug control group showed relief or disappearance of eyelid swelling,no tearing,mucous discharge,and reduced eyelid congestion.The eyes were opened naturally without strain.Compared with the blank group,the ocular symptom scores of mice in the model group and the negative control group increased significantly(P<0.05),and compared with the model group and the negative control group,the ocular symptom scores of mice in the positive drug control group and each dose of The Septiherb Alleviating Allergic Conjunctivitis decreased significantly(P<0.05),and there was no significant difference between the model group and the negative control group(P>0.05).2.Conjunctival and eyelid specimens after HE and toluidine blue staining were observed under light microscope:the conjunctival tissue of mice in the blank group was intact,no edema or thickening was seen,the conjunctival epithelium was neatly arranged,no inflammatory cell infiltration was seen in the conjunctival epithelium and lamina propria,mast cells were occasionally seen,and there was no obvious degranulation.In the model group and the negative control group,the conjunctiva was edematous and thickened,and a large number of inflammatory cells were seen in the conjunctival epithelium and lamina propria,and mast cells were diffuse,and degranulation was obvious.Compared with the model group and the negative control group,the low-dose group,the middle-dose group,the high-dose group and the positive drug control group could reduce the conjunctival edema,the number of inflammatory cells and the degranulation of mast cells to a certain extent,among which,the improvement was more obvious in the middle-dose group,the high-dose group and the positive drug control group.3.ELIS A results:compared with the blank group,the expression levels of serum OVA-sIgE,HIS,TNF-α and IL-4 in the model group and the negative control group were significantly increased,and the expression level of IFN-γ was significantly decreased(P<0.05);compared with the model group and the negative control group,the expression levels of serum OVA-sIgE,HIS,TNF-α and IL-4 in the positive drug control group and each dose group of The Septiherb Alleviating Allergic Conjunctivitis were significantly increased,and the expression level of IFN-y in the high dose group of The Septiherb Alleviating Allergic Conjunctivitis was increased(P<0.05).The expression levels of OVA-sIgE,HIS,TNF-α and IL-4 were reduced in the positive drug control group and the negative control group,and the expression level of IFN-γ was increased in the high dose group of The Septiherb Alleviating Allergic Conjunctivitis(P<0.05).Compared with the positive drug control group,there was no significant difference in the expression levels of serum TNF-α,IFN-γand IL-4 in The Septiherb Alleviating Allergic Conjunctivitis high-dose group,which was not statistically significant(P>0.05),and there was no significant difference in the expression levels of serum OVA-sIgE,HIS,TNF-α,IL-4 and IFN-γ between the model group and the negative control group(P>0.05).4.Western Blot:The relative expression of ERK protein was not statistically significant among the groups(P>0.05);compared with the blank group,the expression level of P-ERK protein in the conjunctival tissues of mice in the model and negative control groups increased significantly(P<0.05);compared with the model group,the expression level of P-ERK protein in the conjunctival tissues of mice in the positive drug control group and each dose group of The Septiherb Alleviating Allergic Conjunctivitis decreased(P<0.05).The expression levels of P-ERK protein in the conjunctival tissues of the mice in the positive drug control group and in the dose groups of The Septiherb Alleviating Allergic Conjunctivitis were not statistically different from those in the positive drug control group(P>0.05);the expression levels of P-ERK protein in the conjunctival tissues of the mice in the low dose group and the high dose group of Chinese medicine were statistically different(P<0.05);the expression levels of P-ERK protein in the conjunctival tissues of the mice in the model group and the negative control group were statistically different from those in the negative control group(P<0.05).There was no significant difference in the expression level of P-ERK protein in conjunctival tissues between the model group and the negative control group(P>0.05).The relative expression of JNK protein was not statistically significant among the groups(P>0.05);compared with the blank group,the expression levels of P-JNK protein in the conjunctival tissues of mice in the model group,the negative control group,the low-dose group of Chinese medicine and the medium-dose group of Chinese medicine increased significantly(P<0.05);compared with the model group,the expression water of P-JNK protein in the conjunctival tissues of mice in the high-dose group of Chinese medicine decreased significantly(P<0.05)The expression level of P-JNK protein in the conjunctival tissue of mice in the high-dose group of Chinese medicine was significantly decreased compared with the positive drug control group(P<0.05),and there was a statistical difference in the expression level of P-JNK protein in the conjunctival tissue of mice in the high-dose group of Chinese medicine compared with the low-dose group of Chinese medicine and the medium-dose group of Chinese medicine(P<0.05),and there was no significant difference in the expression level of P-JNK in the conjunctival tissue of mice between the model group and the negative control group(P<0.05).There was no significant difference in P-JNK protein expression levels between the model group and the negative control group(P>0.05).The relative expression of P38 protein was not statistically significant among the groups(P>0.05),but compared with the blank group,the expression level of P-P38 protein in the conjunctival tissues of mice in each group increased significantly(P<0.05);compared with the model group,the expression level of P-P38 protein in the conjunctival tissues of mice in the low-dose group and the middle-dose group of The Septiherb Alleviating Allergic Conjunctivitis decreased(P<0.05),and the expression level of P-P38 protein in the negative control group increased significantly(P<0.05).The expression level of P-P38 protein in the conjunctival tissues of mice in the negative control group was significantly increased(P<0.05);compared with the positive drug control group,the expression level of P-P38 protein in the conjunctival tissues of mice in the middle dose group of The Septiherb Alleviating Allergic Conjunctivitis was significantly decreased(P<0.05).Conclusion1.Administered by gavage,The Septiherb Alleviating Allergic Conjunctivitis can alleviate allergic symptoms,reduce conjunctival injury,protect conjunctiva and eyelid morphological structure,and reduce mast cell degranulation in AC model mice2.The Septiherb Alleviating Allergic Conjunctivitis can inhibit the release of inflammatory factors HIS,OVA-IgE,IL-4 and TNF-α,promote IFN-y expression and regulate Th1/Th2 balance in AC model mice3.The anti-inflammatory immune effect of The Septiherb Alleviating Allergic Conjunctivitis on AC may be related to the inhibition of inflammatory response caused by the activation of MAPK/ERK/JNK pathway,as it can down-regulate the phosphorylation levels of ERK and JNK proteins related to MAPK signaling pathway... |
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