Renin-Angiotensin System Contributes To The Severity Of Hand,Foot And Mouth Disease

Background:Hand,Foot and Mouth Disease(HFMD)is a common acute infectious disease.Besides,it is generally common in infants and children under 5 years old,and most of the cases presents only mild,while some cases may develop into severe HFMD with severe complications,including encephalitis,meningitis,acute flaccid paralysis(AFP),neurogenic pulmonary edema(NPE),and even death.High death and disability rates of severe HFMD,which seriously endangers the life and health of infants and young children,have become a serious public health problem that needs to be studied and solved urgently.Enterovirus A71(EV-A71)is the main pathogen that appears to induce severe HFMD.Severe HFMD cases are often accompanied by hypertension,renin-angiotensin system(RAS)is an important blood pressure regulation system,and RAS has been proven to be involved in the pathogenesis of a variety of acute infectious diseases,such as severe acute respiratory syndrome(SARS),Coronavirus disease 2019(COVID-19)and avian influenza.Similar to these acute diseases,severe HFMD is often associated with lung inflammation,congestion,and respiratory failure.In order to explore whether RAS is involved in the exacerbation process of HFMD,this study constructed an animal model on the basis of population epidemiology,aiming to reveal the role and possible mechanism of RAS in the exacerbation process of HFMD.Objective:1.To explore whether RAS is related to the severe HFMD.2.To reveal the role and mechanism of RAS in the exacerbation process of HFMD.Methods:1.A case-control study design was adopted to collect the clinical data and relevant examination results of 146 HFMD cases hospitalized in Zhengzhou Children's Hospital from April 2013 to June 2013.According to the clinical diagnostic criteria,the cases were divided into mild group(n=91)and severe group(n=55),with mild group as the control group and severe group as the case group.The basic demographic characteristics,clinical signs,blood routine and biochemical test results of the two groups of patients were collected.The above data were analyzed to explore the factors related to the incidence of severe HFMD.2.Nested serial case-control study design was adopted to collect serum of 30heathy children and 132 HFMD cases hospitalized in the Zhengzhou Children's Hospital from June 2015 to August 2015.According to the clinical diagnostic criteria,132 HFMD cases were divided into mild group(n=79)and severe group(n=53),and30 children were used as the control group.According to the days from the onset of rash or fever to admission,the HFMD cases were divided into 5 subgroups as follow:1,2,3,4,and 5 days,respectively named?,?,?,?and?.Enzyme-linked immunosorbent assay(ELISA)was used to detect the concentrations of Angiotensin?(Ang?)and Norepinephrine(NE),to explore whether the RAS was involved in the pathogenesis of severe HFMD.3.The animal model of HFMD was established by intraperitoneal injection of EV-A71,and the mice in the control group were injected with cell culture supernatant with the same volume,and clinical symptoms were observed day by day.Mice were sacrificed at different times after infection.Serum of mice was collected and the levels of inflammatory factors and molecules associated with RAS were measured.the brain and lung of mice were taken for pathological examination,and RAS related gene mRNA expression levels were measured by q RT-PCR,to reveal whether RAS was involved in the exacerbation process of HFMD.4.Angiotensin ? type 1 receptor blockers(ARBs)and Angiotensin converting enzyme inhibitors(ACEI)were used for the intervention of animal model of EV-A71infection,the clinical symptoms were observed day by day.Mice were sacrificed at different times after infection.Serum of mice was collected and the levels of inflammatory factors were measured.the brain and lung of mice were taken for pathological examination,and inflammatory related gene mRNA expression levels were measured by q RT-PCR,to reveal the role and mechanism of RAS in the exacerbation process of HFMD.Results:1.The levels of T lymphocyte(T cell,CD3~+),helper T cell(Th cell,CD3~+CD4~+)and Th cell/Ts cell(CD4/CD8)in the severe group were lower than those in the mild group,with statistically significant differences(P<0.05).The levels of natural killer cells(NK cell,CD16~+CD56~+)and B lymphocyte(B cell,CD19~+)in severe group were higher than those in the mild group,with statistically significant differences(P<0.05).The levels of cytotoxic T cells(CTL,CD3~+CD8~+)in the severe group were lower than that in the mild group,with no statistically statistical differences(P>0.05).Complement(C)3,C4,and Immunoglobulin(Ig)A levels were higher in the severe group than those in the mild group,with statistically significant differences(P<0.05).Ig M and Ig G levels in the severe group were higher than those in the mild group,with no statistically statistical differences(P>0.05).Diastolic blood pressure,systolic blood pressure,respiratory rate,and rate of abnormal breathing(long sighing or double inspiration)were higher in the severe group than those in the mild group,with statistically significant differences(P<0.05).The pulse rate of patients in the severe group was higher than those in the mild group,with no statistically statistical differences(P>0.05).2.The Ang ? and NE concentrations of HFMD patients(mild or severe)were higher than those in healthy control group,with statistically significant differences(P<0.05).The Ang?concentrations were higher in the severe group than those in mild group,with statistically significant differences(P<0.05).The Ang?and NE concentrations of severe HFMD cases peaked on the 3rd day after onset.Ang?and NE concentrations were positively correlated(mild group:r=0.492,P<0.001;severe group:r=0.645,P<0.001).there was no statistically significant difference among the concentrations of Ang?and NE,respectively,during 1st day to 5th day after the onset,P>0.05.During 1st day to 5th day after the onset,Ang?concentrations were higher in the severe group than those in mild group,with statistically significant differences(P<0.05).During the 2nd day to 4th day after onset,NE levels in the severe group were higher than those in the mild group,with statistically significant differences(P<0.05).Both of Ang?and NE concentrations in the severe group increased at the 1st three days after on set and decreased after peaking on the 3rd day.3.No significant pathological changes in brain or lung of mice were found in the control group from 1 day(s)post infection(dpi)to 5 dpi.At 3 dpi,the brain lesions of mice infected with EV-A71 were more severe than those in the control group,with statistically significant differences in pathological scores between the two groups(P<0.05).During 3?5 dpi,EV-A71-infected mice showed more lung lesions,such as alveolar enlargement and red blood cell leakage,with statistically significant differences in pathological scores between the two groups(P<0.05).During 1?5 dpi,the interleukin(IL)-6,IL-10,Ang?,Ang(1-7),Angiotensin converting enzyme(ACE)and ACE2 concentrations of EV-A71-infected mice increased,compared with the control mice with statistically significant differences(P<0.05).During 1?3 dpi,the tumor necrosis factor(TNF)-?concentrations of EV-A71-infected mice were higher than that of mice in the control group,with statistically significant differences(P<0.05).At 3 dpi and 5 dpi,the interferon(IFN)-?concentrations of mice in the EV-A71 infected group were higher than that of mice in the control group,with statistically significant differences(P<0.05).The ratio of Ang?and Ang(1-7)concentrations in the EV-A71-infected group at 3 dpi and 4 dpi was higher than the control group,with statistically significant differences(P<0.05).During 1?5 dpi,there was no statistically significant difference of average optical density(AOD)change of platelet endothelial cell adhesion molecules(PECAM-1/CD31)and Angiotensin?type 1 receptor(AT1R)in the brain and lung of mice in the control group(P<0.05).The AOD of AT1R in brain in EV-A71infected group was higher than that in control group during 1?5 dpi(P<0.05).During3?5 dpi,the AOD of CD31 in brain in the EV-A71 infected group was higher than that in the control group(P<0.05).During 3?5 dpi,the AOD of CD31 and AT1R in the lung of EV-A71-infected mice were higher than those of control group(P<0.05).During 1?5 dpi,the expression of Renin and Angiotensinogen(AGT)mRNA of EV-A71-infected mice were higher than that of mice in control group(P<0.05).During 1?3 dpi,the expression of ACE,Mas receptor(Mas R)and vascular endothelial growth factor(VEGF)mRNA of EV-A71 infected mice were higher than that of mice in the control group,with statistically significant differences(P<0.05).The mRNA expression of Renin,AGT,ACE and Mas R in the lung of mice infected with EV-A71 decreased at 2 dpi compared with that of 1 dpi(P<0.05),increased to the peak at 3 dpi value(P<0.05),began to decrease at 4 dpi(P<0.05).AT1R mRNA expression in lung of EV-A71-infected mice was lower than that of mice in the control group at the early stage of infection(1?2 dpi)(P<0.05).The mRNA expression of AT1R and VEGF in lung of EV-A71-infected mice were continuously increased during 3?5 dpi(P<0.05).4.ARBs(irbesartan,30 mg/kg.bw)and ACEI(captopril,90 mg/kg.bw)were used for intervene in EV-A71-infected mice at 3 dpi.The clinical scores,the pathological scores of brain and lung and the AOD of CD31 and AT1R in brain and lung of mice were lower in ARBs and ACEI intervention group than that in EV-A71group(P<0.05).At 5 dpi,IL-6 concentration in ARBs intervention group was lower than that in EV-A71 group and control group,with the statistically significant differences(P<0.05).The IL-6,IL-10 and IFN-?concentrations in ARBs and ACEI intervention groups were significantly lower than those in EV-A71 group(P<0.05).At the 5 dpi,the mRNA expression of IL-6,IL-10,TNF-?and IFN-?in brain and lung of mice in ARBs and ACEI intervention groups were all lower than those in the EV-A71 group,with statistically significant differences(P<0.05).The mRNA expression of IL-6 in the brain in the ARBs and ACEI intervention group was higher than that of the control group,with statistically significant differences(P<0.05).The mRNA expression of TNF-?in the ACEI intervention group was higher than that in the control group(P<0.05).The mRNA expression of IL-6,IL-10,TNF-?and IFN-?in the lung of mice in the ARBs and ACEI intervention groups were higher than those in the control group,with statistically significant differences(P<0.05).Conclusion:1.Increased blood pressure was associated with severe HFMD,and the activation of RAS was involved in the exacerbation process of HFMD.2.EV-A71 infection could activate the RAS in mice,RAS inhibitor intervention could reduce the inflammatory response and block the exacerbation of EV-A71-infected mice.