RMK-03^(TM)皮肤黏膜护理喷剂对上呼吸道黏膜免疫功能低下小鼠免疫调节机制的影响

Influence of RMK-03^(TM) Skin and Mucous Membrane Spray on Immune Regulation Mechanism in Mice with Upper Respiratory Mucosal Immune Dysfunction

目的:通过寒冷致小鼠上呼吸道黏膜免疫功能低下(upper airway immunity dysfunction,UAID)模型的实验,基于免疫球蛋白A单克隆抗体(immunoglobulin A monoclonal antibody,IgA MAb)阻断新型冠状病毒(SARSCoV-2)的Spike-ACE 2相互作用探究RMK-03^(TM)喷剂对上呼吸道黏膜的免疫调节机制。方法:将40只SPF级ICR小鼠分为正常组、UAID模型组、利巴韦林喷雾剂组、RMK-03;喷雾剂组4组,每组10只;利巴韦林喷剂组和RMK-03^(TM)喷剂组先给予口腔喷雾0.1 mL/只q12h,给药3 d,;第3 d,除正常组外其他各组小鼠复制UAID模型,即将小鼠放入-20℃寒冷环境中15 min,之后在室温适应1 h,经腹腔注射毛果芸香碱滴眼液(0.25 mL/只),2 min后以安乐处死小鼠,取口腔、咽喉部黏膜组织进行HE病理学切片检查,以Western blotting法考察其IgA、pIgR蛋白的表达,采用ELISA法检测细胞因子IL-4、IL-6和IFN-γ、TNF-α含量;采用免疫组化法检测ACE 2和ACE的表达情况。结果:UAID模型组小鼠唾液中SIgA含量显著低于正常组(P<0.05),提示模型复制成功;与正常组相比,UAID模型组小鼠的口腔和咽喉黏膜组织可见轻度间质水肿、小血管扩张充血,中性粒细胞伴有淋巴管轻度扩张水肿;UAID模型组小鼠口腔、咽喉部黏膜组织IgA、pIgR蛋白表达,IgA相关细胞因子IL-4、IL-6的含量及pIgR相关细胞因子IFN-γ、TNF-α的含量均显著低于正常组(P<0.05),该组的ACE、ACE 2平均光密度值(IOD/area)均显著性高于正常组(P<0.05);利巴韦林喷剂组和RMK-03;喷剂组的上述主要指标均在不同程度上优于UAID模型组,且后者的上述主要指标优于前者。结论:RMK-03^(TM)皮肤黏膜护理喷剂通过调节局部黏膜免疫屏障低下相关IgA的含量,抑制呼吸道黏膜中ACE 2活性表达,间接推测其阻断SARS-CoV-2的Spike-ACE 2相互作用以提供黏膜免疫。

Objective: To explore the immunomodulation mechanism of RMK-03^(TM) spray on upper respiratory mucosa based on immunoglobulin A(IgA MAb) blocking Spike-ACE 2 interaction of novel Coronavirus(SARS-CoV-2)in the mice experiment of the upper airway mucosal immune dysfunction(UAID) model induced by cold. Methods:40 SPF ICR mice were divided into 4 groups, including normal group, UAID Model group, ribavirin spray group and RMK-03^(TM) spray group, with 10 mice in each group. Ribavirin spray group and RMK-03^(TM) spray group were given oral spray 0.1 mL/mouse q12 h for 3 days. On day 3, other groups except normal group were used to replicate UAID model.The mice were placed in a cold environment of-20℃ for 15 min, and then acclimated to room temperature for 1 h.The mice were intraperitoneally injected with pilocarpine eye drops(0.25 mL/mouse). The expression of IgA and pIgR proteins were examined by Western blotting method. The contents of cytokines IL-4 and IL-6, IFN-γ and TNF-αwere detected by ELISA method. The expression of ACE 2 and ACE were detected by immunohistochemistry. Results:The content of SIgA in saliva of mice in UAID model group was significantly lower than that in normal group(P<0.05), suggesting that the model was successfully replicated. Compared with normal group, UAID model group showed mild interstitial edema, dilatation and congestion of small vessels, and neutrophils accompanied by mild dilatation and edema of lymphatic vessels. The expression of IgA and pIgR proteins in oral and pharyngeal mucosa of mice in UAID model group, the contents of IgA-related cytokines IL-4 and IL-6, and the contents of pIgR-related cytokines IFN-γ and TNF-α were significantly lower than those in normal group(P<0.05). The mean optical density(IOD/area) of ACE and ACE 2 in the group were significantly higher than that in the normal group(P<0.05). The above main indexes of ribavirin spray group and RMK-03;spray group were better than UAID model group to varying degrees, and the above main indexes of ribavirin spray group were better than UAID model group. Conclusions: RMK-03^(TM) skin and mucosal care spray inhibits the active expression of ACE 2 in respiratory mucosa by regulating the content of IgA associated with low local mucosal immune barrier, and it is indirectly speculated that RMK-03^(TM) can block the spike-ACE 2 interaction of SARS-COV-2 to provide mucosal immunity.