Schistosoma Japonicum-derived Peptide SJMHE1 Ameliorates Allergic Symptoms And Responses In Mice With Allergic Rhinitis

Objective:Allergic rhinitis（AR）is a common chronic inflammatory disease,affecting people’s quality of life,work efficiency,and educational performance seriously,and has become a serious global public health problem.Type-2 immune environment is the fundamental element of AR,which induces immunoglobulin E（Ig E）produce and results in the appearance of symptoms such as pruritus,rhinorrhoea,nasal congestion rubbing and sneezing.Antihistamines,corticosteroids and antileukotrienes are effective for the symptom management of AR patients,however,they cannot induce the disease regression.Therefore,there is still a need to discover new and effective therapeutic drugs.Helminths and their products have shown efficacy in preventing allergic and autoimmune diseases in a variety of mouse models.SJMHE1,a small molecular peptide derived from the heat shock protein 60（HSP60）of Schistosoma japonicum,is reported to alleviate airway inflammation in allergic asthma mice.In this study,the potential intervention effect and mechanisms of SJMHE1 in allergic rhinitis（AR）was investigated.Methods:1.20 female BALB/c mice,6-8 weeks,were randomly divided into four groups:Control,PBS,OVA323-339 and SJMHE1 groups.A mouse model of allergic rhinitis was established using ovalbumin（OVA）,and mice were sensitized by intraperitoneal injection of OVA（10μg）and 2 mg of Al（OH）₃in 200μL PBS at day 0,7,and 14,respectively.Subsequently,at day 21-27 and 29-35,mice were given intranasal infusion of OVA（100μg）for stimulation.At day 0,14,and 28,mice in the PBS,OVA^323-339,and SJMHE1 groups were separately given subcutaneous injections of100μL PBS,OVA^323-339,or SJMHE1（10μg,emulsified with 45μL incomplete Freund’s adjuvant and 45μL PBS）.On day 36,all mice were killed and samples collected.2.On the 35th day,the number of mice rubbing and sneezing was recorded immediately within 20 minutes after the last nasal challenge.Take representative images of skin lesions on the nose of mice.3.HE,Wright-Giemsa,and Masson trichrome staining were used to evaluate the inflammatory response,eosinophil infiltration,and fibrosis of the mouse nasal mucosa.4.The nasal of mice was irrigated with PBS,and the nasal lavage fluid was collected.The levels of IL-4,IL-13,and IL-10 in the nasal lavage fluid of mice were detected by ELISA.The expressions of IL-4,IL-13,and IL-10 m RNA in mice spleen were detected by q PCR.The levels of IL-4,IL-13,IL-10,and OVA specific Ig E in mouse serum were detected by ELISA using mouse eyeball blood.5.The mice were injected subcutaneously with FITC fluorescent labeled SJMHE1.At day 3,7,and 14,the nasal mucosa and spleen of the mice were taken and frozen sections were made.The distribution of FITC-SJMHE1 was detected under a fluorescence microscope.6.The proportion of CD4⁺FITC⁺,CD19⁺FITC⁺,CD11c⁺FITC⁺,and F4/80⁺FITC in the spleen were detected by flow cytometry.Mouse spleen B cells were sorted by flow cytometry for RNA sequencing analysis.The proportion of Bregs and B10 cells in the spleen of mice in each group was further analyzed.Western blot was used to detect the expression of Prdm1 in the spleen of mice in each group.7.Culture B cells in vitro:immunofluorescence analysis was performed to determine whether SJMHE1 binds to B cells.Flow cytometry was used to analyze the effect of SJMHE1 on Bregs differentiation.8.Analyze the safety of subcutaneous injection of SJMHE1 was analyzed.HE staining was used to evaluate the pathological changes of the main organs（heart,liver,spleen,lung,and kidney）in mice.The body weight changes of normal mice and SJMHE1 treated mice were compared.Serum indicators（ALT,AST,UREA,and CREA）were used to evaluate the liver and kidney functions of mice.Results:1.Compared with the control group,mice in the PBS group and OVA^323-339group experienced significant nasal symptoms（rubbing and sneezing）and skin damage to the nose after nasal stimulation.The symptoms of rubbing and sneezing,as well as the degree of skin damage to the nose in the SJMHE1 group were significantly reduced compared to the PBS group and OVA^323-339group.2.The pathological results of the nasal mucosa showed that compared with the control group,the inflammatory reaction of the nasal mucosa in the PBS group and the OVA^323-339group significantly were increased,and the infiltration of eosinophils was significantly increased.Compared with PBS group and OVA^323-339group,the nasal mucosal inflammatory reaction and eosinophil infiltration in SJMHE1 treated mice were significantly reduced.In the control group,only weak collagen fibers were present in the lamina propria of the nasal mucosa of mice,while in the PBS group and OVA^323-339group,there was significant accumulation of collagen fibers in the basal and lamina propria of the nasal mucosa.Compared with the PBS or OVA ^323-339groups,the accumulation of collagen fibers in the basal and lamina propria of the SJMHE1 group was significantly reduced.3.Compared with the control group,the levels of IL-4 and IL-13 in the nasal lavage fluid of mice in the PBS group and the OVA^323-339group were significantly increased,while there was no significant change in IL-10.Compared with PBS group and OVA^323-339group,the nasal lavage fluid of SJMHE1 group mice significantly decreased IL-4 and IL-13,and significantly increased IL-10.The results of q PCR showed that compared with the control group,the expressions of IL-4 and IL-13 in the spleen of mice in the PBS group and OVA^323-339group were significantly increased,while the expression of IL-10 was significantly decreased.Compared with PBS group and OVA^323-339group,the expressions of IL-4 and IL-13 in SJMHE1group were significantly decreased,while the expression of IL-10 was significantly increased.The results of serum ELISA showed that compared with the control group,the serum levels of IL-4,IL-13,and OVA specific Ig E in PBS group and OVA^323-339group were significantly increased,while there was no significant change in IL-10.Compared with PBS group and OVA^323-339group,serum levels of IL-4,IL-13,and OVA specific Ig E in SJMHE1 group were significantly down-regulated,while IL-10was significantly up-regulated.4.The immunofluorescence results showed that on days 3,7,and 14,there was no FITC-SJMHE1 distribution in the mice nasal mucosa,while on days 3,7,and 14,FITC-SJMHE1 fluorescence was observed in the spleen.5.The results of flow cytometry showed that the ratios of CD19⁺FITC⁺cells were the highest in the spleen compared to CD4⁺FITC⁺,CD11c⁺FITC⁺and F4/80⁺FITC cells.RNA sequencing results showed that the Hspa1a and Hspa1b genes in B cells of SJMHE1 group were significantly up-regulated compared to the control group.Compared with the control group,the proportions of Bregs and B10 cells in the spleen of mice in the PBS group were significantly decreased,while the proportions of Bregs and B10 cells in the SJMHE1 group were significantly increased compared to the PBS group.Western blot results showed that the expression of Prdm1 in the spleen of mice in PBS group was down-regulated compared to the Control group;After the SJMHE1 treatment,the expression of Prdm1 was up-regulated.6.Culture B cells in vitro:Immunofluorescence showed that SJMHE1 bound to B cells;Flow cytometry analysis showed that SJMHE1 up-regulated the ratio of Bregs.7.The pathological results showed that compared with the control group,the heart,liver,spleen,lungs,and kidneys of the SJMHE1 group had no significant pathological changes.The body weight of the SJMHE1 group mice did not significantly change.There were no significant changes in serological indicators（ALT,AST,UREA,and CREA）.Conclusions:1.SJMHE1 ameliorated the allergic symptoms and reactions in OVA-induced allergic rhinitis mice.2.SJMHE1 reached the spleen and regulated the immune response of the system.3.SJMHE1 promoted the up-regulation of Bregs and B10 cells in the spleen of mice,which might play a protective role in allergic rhinitis.4.This study partially confirmed the safety of SJMHE1 administration.Our findings may provide a new approach for the treatment of allergic rhinitis.