Pharmacodynamics And Mechanism Of Dimethyl Sulfone Screened From Urine Of COPD Patients And Healthy Volunteers

The incidence of chronic obstructive pulmonary disease(COPD)in the world over 40 years old is 9%-10%,and the incidence rate in China is about 8.2%.The main manifestation of COPD is the incomplete reversible airflow limitation,and the severity of the disease is often accompanied by an abnormal inflammatory response.The incidence of COPD is higher in older people due to decreased body function and decreased immune function.Glucocorticoids are often used clinically for the symptoms of inflammation in COPD patients.However,these drugs produce adverse reactions such as osteoporosis and anti-immune function.Therefore,it is of great clinical significance to find potential drugs that can alleviate the low immunity associated with COPD patients and improve the immune function of COPD patients.In this paper,we screened potential drugs for the prevention and treatment of COPD patients in the urine of COPD patients and healthy volunteers,and explored the pharmacological pharmacodynamics and mechanism of the drug's immune and anti-inflammatory effects.This paper mainly studies through the following four chapters:Part ?: We screened differentially expressed small molecules from the urine of COPD patients and healthy volunteers,and then found potential drugs that mediate immunity and prevent COPD patients.We first used GC-MS analysis technology to detect the urine of COPD patients and healthy volunteers,so as to study the differential expression and regularity of metabolic small molecules between the two groups.The aim is to discover new biomarkers for the diagnosis of COPD,or to screen for small molecules of endogenous metabolism that prevent COPD.Specific methods were as follows: morning urine was collected from 35 healthyadults and 36 COPD patients,respectively.After 50% methanol water extraction,centrifugation,filtration and other sample pretreatment,The conditions of mass spectrometry such as inlet temperature 200 ?,FID detector temperature 200 ?,EI ionization,full scan range(m/z): 50-650 were analyzed.The above specimens were detected by GC-MS,and the GC-MS spectra of each sample were analyzed by SIMCA14.1 analysis software to find out the differentially significant small molecular metabolic markers.The experimental results show that there are 9 metabolites with significant differential expression in the urine of COPD patients and healthy volunteers.which are:2-oxabicyclo[2.2.2]octan-6-ol,1,3,3-trimethyl-,nicotinamide,indole,benzoamide,benzoic acid,hydroxybenzene,phenylacetic acid,methyl sulfonyl methane,1-methyl-4(1-methylethenyl)-Benzene.Based on literature review and preliminary experimental results,MSM with high expression in urine of healthy volunteers was selected for systematic pharmacological study.The retention time was 20.432 min.After comparison with the NIST 14.0 mass spectrometer standard library,it was found that the corresponding substance of the peak had a matching degree with dimethyl sulfone of 96%,and it was confirmed to be dimethyl sulfone.Part ? : This section is to investigate the pharmacological pharmacodynamic effects of MSM on LPS-induced inflammation in mice.We first established a mouse model of LPS-induced inflammation and analyzed the effect of MSM on serum inflammatory factors IL-1?,TNF-? and IL-6 by ELISA kit analysis.We used HE staining to observe the effect of MSM on lung tissue,and observed the effect of MSM on immune function in mice through thymus coefficient,spleen coefficient and mouse carbon clearance test.(1)The levels of IL-1?,IL-6 and TNF-? in the MSM group showed a downward trend,and the decreasing trend was dose-dependent.The MSM drug group at 100,300,and 900 mg/kg decreased the expression of TNF-? by 12.3%(P<0.01),12.9%(P<0.01),and 17.0%(P<0.01),respectively.The expression levels of IL-6 were down-regulated by 6.3%,20.5%,and 24.2%,respectively(P<0.05).The expression level of IL-1? was down-regulated by 4.5%,6.2%,and 20.1%,respectively.The study also found that the 900 mg/kg MSM dose group had stronger inhibition of TNF-? than the DXMS positive group.(2)HE staining results showed that MSM had protective effects on lung tissue of LPS-induced inflammatory mice.And with the increase of MSM dose,the alveolar wall of mice was significantly thinner,and the infiltration of macrophages and neutrophils was alleviated.(3)MSM did not affect the thymus coefficient and spleen coefficient of mice.The thymus coefficient and spleen coefficient of MSM group had no significant difference compared with the normal group,and the mouse carbon clearance experiment showed that MSM has the function of improving immune function..Part ?: To study the pharmacological and pharmacodynamic effects of MSM on imiquimod-induced psoriasis-like mice.By establishing imiquimod-induced psoriasis-like mice model,the effects of MSM on serum inflammatory factors IL-17 F,IL-23 and TNF-alpha were detected.PASI score was used to evaluate the pharmacological effects of MSM on psoriasis-like mice.HE staining was used to observe pathological changes and immunohistochemistry was used to further explore the effects of MSM on IL-17 F,IL-23,TNF-a,JAK2,STAT3 and NF-? b.(1)In imiquimod-induced psoriasis-like mice model,PASI score preliminarily showed the protective effect of MSM on psoriasis-like mice: cortical thickening was reduced,and the severity of the lesion was also reduced.HE staining further proved that MSM had protective effect on psoriasis-like mice,such as reduced cortical thickness and inflammatory cell infiltration.To study the pharmacodynamic effects of MSM on imiquimod-induced psoriasis-like mice.By establishing imiquimod-induced psoriasis-like mice model,PASI score and HE stainingconfirmed the success of the model.To detect the effects of MSM on serum inflammatory cytokines IL-17 F,IL-23 and TNF-? in mice of each group.Preliminary evaluation of the pharmacological effects of MSM on the lesions of psoriasis mice by PASI score,The pathological changes and signs were observed by HE staining.Immunohistochemistry was used to further investigate the effects of MSM on antibodies IL-17 F,IL-23,TNF-? and JAK2,STAT3 and NF-? b.(1)In imiquimod-induced psoriasis-like mice model,PASI score preliminarily showed the protective effect of MSM on psoriasis-like mice: cortical thickening was reduced,and the severity of the lesion was also reduced.HE staining further proved that MSM had protective effect on psoriasis-like mice,such as reduced cortical thickness and Neutrophils and macrophages cell infiltration..(2)The serum levels of IL-17 F and IL-23 in the MSM administration group were significantly inhibited compared with the model group.The serum levels of TNF-? in the MSM medium and high dose groups were lower than those in the model group.The inhibition rates of IL-17 F in the MSM group at 100 mg/kg,300 mg/kg,and 900 mg/kg were 80.9%(P<0.01),63.8%(P<0.01),and 59.6%,respectively(P<0.01).).The inhibition rates of IL-23 were 13.8%,13.1%,and 12.5%,respectively,but there was no significant difference.The inhibition rates of TNF-? in the MSM group at 300 mg/kg and 900 mg/kg were 18.8% and 34.5%,respectively(P <0.01).(3)The results of immunohistochemistry further confirmed that MSM significantly inhibited the expression of IL-17 F,IL-23,TNF-?,JAK2,STAT3 and NF-? b(all P values less than 0.01).The most significant of these was the effect on STAT3 protein,which produced a two-way regulation,ie,a small dose(100 mg/kg)was up-regulated,and a medium-high dose(300,900 mg/kg)down-regulated the expression of STAT3 protein.Part ?: Molecular mechanism of immune regulation of MSM.MTT assay was used to detect the cytotoxicity of MSM and determine thenon-toxic dose.Then the mechanism of immune regulation of MSM was studied by Western Blot method,and the MAPK signal transduction pathway was selected for analysis.(1)MTT assay showed that 12.5,25 and 50?mol/L MSM had no cytotoxic effect on MRC-5 and RAW264.7cells.(2)LPS stimulated RAW264.7 cells could significantly increase the phosphorylation levels of p38,ERK and JNK in the MAPK pathway.MSM inhibited phosphorylated p38,ERK and JNK in varying degrees(P< 0.05),and the inhibition was dose-dependent,but there was no significant difference in the up-regulation of p38,ERK and JNK compared with the model group.In conclusion,the dimethyl maple,a substance with significant difference between healthy people and COPD patients,was successfully detected by GC-MS technology,and its function was preliminarily studied.The results suggest that MSM has significant anti-inflammatory activity,which may be related to the reduction of the expression of inflammatory factors IL-1??IL-6?IL-17F?IL-23?TNF-? inhibition of the functional expression of JAK2,STAT3 and NF-?b?p-p38,p-ERK,p-JNK in the MAPK pathway and enhancement of immune function.Further large sample studies will confirm whether MSM is a small molecular marker for anti-inflammatory and regulating immune balance to detect COPD,and lay a theoretical foundation for the development of new immunomodulator to prevent and treat COPD.