| Objective: Mahuang Xixin Fuzi Decoction（MXF）is a classical prescription for the treatment of two sensations of deficiency of kidney-yang.Previous studies have confirmed that MXF has a good intervention effect on the exogenous mice model of kidney-yang deficiency.In this study,the pharmacodynamic effect and mechanism of the polysaccharide of Mahuang Xixin Fuzi Decoction（MXF polysaccharide）on the exogenous mice model of kidney-yang deficiency were studied,and to explore the pharmacodynamic material basis and mechanism of MXF in the treatment of influenza diseases.Methods: 1.Compound polysaccharides,ephedra polysaccharides,Asarum polysaccharides and aconite polysaccharides from MXF（Ephedra: Asarum: Aconite,3:3:6）were extracted by water extraction and ethanol precipitation.The content of polysaccharides was determined by phenol sulfuric acid method,protein content was determined by Coomassie brilliant blue method,and monosaccharide composition of Ephedra polysaccharides,Asarum polysaccharides,aconite polysaccharides and MXF polysaccharides was determined by gas chromatography.2.Screening of Housekeeping Genes in Lung Tissues of Kidney-Yang Deficiency Syndrome Mice Model Infected with Influenza Virus A: Balb/c mice（10 mice）were intraperitoneally injected with 2 mg/m L estradiol benzoate at 4 m L/kg once a day for 7 days.The kidney-yang deficiency model was replicated.Autonomous activity,rectal temperature and swimming time were measured to determine the success of the model.Another 10 Balb/c mouse were selected as normal mice and injected with the same amount of saline.Then infected with influenza A virus by nasal（TCID50 2.34×108）,and the normal group was dripped with the same amount of saline.The mice were fed in isolation after nasal drip infection.After 7 days of observation,the mice were sacrificed and the lung tissues were taken to calculate the organ index.Real-Time PCR was used to detect the expression stability of 15 reference genes in lung tissues,and ge Norm,Norm Finder and Bestkeeper methods were used to evaluate 15 reference genes.3.Pharmacodynamics of MXF Polysaccharide Anti-influenza Virus in Vivo:（1）There were 70 male Balb/c mice,10 of which were in the normal group.The other mice were intraperitoneally injected with 2 mg/m L estradiol benzoate at 4 m L/kg once a day for 7 days to replicate the kidney yang deficiency model.H1N1 influenza virus nose dropping infection were replicated.The model group randomly divided into normal exogenous model group,normal exogenous MXF group,normal exogenous MXF polysaccharide group,exogenous model group of kidney-yang deficiency,exogenous MXF group of kidney-yang deficiency and exogenous MXF polysaccharide group according to body weight and rectal temperature.MXF and MXF polysaccharide were administered by gavage respectively,and the dosage of MXF was 1.8 g·kg-1·d-1,and the dosage of MXF polysaccharide was 65 mg·kg-1·d-1,normal group is fed with physiological saline of equal volume,once a day for 6 days,body weight and Anal temperature are measured daily;after dissection,lung,thymus,spleen,liver,kidney,adrenal gland,seminal vesicle gland,testis are taken,organ index is calculated,and partial least squares discriminant analysis was used to analyze and compare the pharmacodynamic differences between MXF polysaccharides and MXF prescriptions.（2）There were 80 male Balb/c mice,10 of which were in the normal group.The other mice were intraperitoneally injected with 2 mg/m L estradiol benzoate at 4 m L/kg once a day for 7 days to replicate the kidney yang deficiency model.H1N1 influenza virus nose dropping infection were replicated.The model group randomly divided into model group,MXF group,MXF polysaccharide group,ephedra polysaccharide group,Asarum polysaccharide group,aconite polysaccharide group and MXF depolysaccharide group according to body weight and rectal temperature.The MXF dose was 1.8 g·kg-1·d-1,which was equivalent to human clinical equivalent dose.MXF polysaccharide(120 mg·kg-1·d-1),ephedra polysaccharide(20 mg·kg-1·d-1),aconite polysacc Asarum polysaccharide(73.2 mg·kg-1·d-1),polysaccharide(130 mg·kg-1·d-1),intragastric administration of 0.4 m L daily,normal group and model group were given saline of equal volume for 6 days.After dissection,lung,thymus,spleen,adrenal gland and seminal vesicle were taken to calculate organ index.The number of M gene copies of H1N1 and the relative expression of several anti-inflammatory and pro-inflammatory cytokines in lung tissue were determined by RT-PCR.The expression of multi-cytokines in lung tissue supernatant was determined by flow cytometry CBA.4.Antiviral Activity and Immune Activity of MXF Polysaccharide and Single Drug Polysaccharide in Vitro: CCK8 method was used to study the anti-H1N1 influenza effect of Ephedra polysaccharides,Asarum polysaccharides,aconite polysaccharides and MXF polysaccharides in vitro;MTT method was used to study the immune effects of MXF polysaccharides and mono-drug polysaccharides on spleen cells and peritoneal macrophages of mice.Results: 1.The standard curve of polysaccharide content was Y=0.0135X-0.0314,and the standard curve of protein content was Y=5.0853X-0.0482.The contents of MXF polysaccharide,ephedra polysaccharide,Asarum polysaccharide and Fuzi polysaccharide were 57.48%,11.21%,25.22% and 83.56%,respectively.The protein content was 1.640%,1.560%,0.800% and 1.441% respectively.The monosaccharide composition of eptance of Ephedra polysaccharide was D-arabinose,D-xylose and D-mannose.D-glucose,D-galactose;Asarum polysaccharide monosaccharide composition is L-rhamnose,D-arabinose,D-xylose,D-glucose,D-galactose;Aconitum polysaccharide monosaccharide composition is D-arabinose,D-xylose,D-galactose;MXF polysaccharide monosaccharide composition is D-arabinose,D-xylose,D-glucose,D-galactose.2.Compared with the normal group,the weight and rectal temperature of the model group mice were significantly decreased,the lung index was increased,and the absolute copy number of M gene of H1N1 in lung tissue was more than 6.1×106/ml,which indicated that the model of kidney-yang deficiency exogenous sensation was successfully constructed.The correlation coefficients of 15 reference genes were greater than 0.990,and the amplification efficiency was between 91% and 98.9%,which could be used to evaluate the stability of the reference gene.Best Keeper software analysis showed that the stability of Nedd8 gene was the best,while the stability of β2m gene was the worst;Ge Norm software analysis showed that Ywhea and Tuba genes were the best,while β2m gene was the worst;Norm Finder software analysis showed that the stability of 18 Sr RNA gene was the best,and β2m gene was the worst;The difference of gene expression between normal group and model group was tested by T test.The maximum P value of Grcc10 + Ppia combination was 0.986,and the minimum average difference was 0.009.It can be used as the best choice of housekeeping gene for lung tissue of mice model of Kidney-Yang Deficiency Syndrome Mice Model Infected with Influenza Virus A.3.1（1）It can be seen from the comprehensive efficacy scores of each group of PLS,compared with the normal group,the comprehensive pharmacodynamic scores of the other groups decreased significantly（P<0.01）;compared with the exogenous model group of kidney-yang deficiency,the comprehensive pharmacodynamic scores of the normal exogenous model group,the normal exogenous MXF group and the normal exogenous MXF polysaccharide group increased;the comprehensive pharmacodynamic scores of the exogenous MXF group of kidney-yang deficiency and the exogenous MXF polysaccharide group of kidney-yang higher than that in the model group of kidney-yang deficiency（P<0.01）.（2）The body weight and rectal temperature of mice with kidney-yang deficiency showed a downward trend after infection with the virus.After administration,the body weight and rectal temperature of MXF group and MXF polysaccharide group showed an upward trend,while the relative expression of influenza A virus H1N1 of M gene copies in MXF,MXF polysaccharide group and asarum polysaccharide group decreased significantly（P<0.05）.The gene expression of cytokines was detected,MXF polysaccharide could decrease the relative expression of TNF-α,IL-1α,IL-1β and IL-27 in lung tissue of mice with kidney-yang deficiency exogenous sensation,but there was no significant difference.MXF polysaccharide could significantly reduce the relative expression of IL-6（P<0.05）,MCP-1（P<0.05）,IFN-β（P<0.01）,and MXF polysaccharide could also increase the relative expression of IL-10 and IFN-γ.The contents of IL-10,IL-23,IL-1beta,MCP-1 and IFN-gamma in MXF polysaccharide group were lower than those in MXF group,but there was no significant difference.Asarum polysaccharide could significantly reduce the contents of cytokines in single drug polysaccharide.4.IC50 of Ephedra polysaccharides,Asarum polysaccharides,aconite polysaccharides,MXF polysaccharides and ribavirin against H1N1 influenza virus In vitro were calculated by CCK8 method.IC50 of Ephedra polysaccharides,aconite polysaccharides,ribavirin against MDCK cells were 0.0221 mg/m L,0.008775 mg/m L,0.7516 mg/m L,0.03046 mg/m L,02032 mg/m L,TI values were 4.009,14.24,13.30,13.09,4.208;MXF polysaccharides were 250μg/m L and 15.6μg/m L,respectively.The proliferation of spleen cells and macrophages was the most obvious（P<0.01）.Ephedra polysaccharides,Asarum polysaccharides and aconite polysaccharides at 250ug/m L and 31.25μg/m L（P<0.01）,62.5μg/m L（P<0.05）and 31.25μg/m L（P<0.01）,25μg/m L（P<0.05）and 100μg/m L（P < 0.01）had significant proliferation effects on spleen cells and macrophages,respectively.Conclusion: MXF polysaccharide has a good intervention effect on Kidney-Yang Deficiency Syndrome Mice Model Infected with Influenza Virus A.MXF polysaccharides and asarum polysaccharides can reduce lung index and H1N1 expression in lung tissue of mice with kidney-yang deficiency exogenous sensation.And significantly reduced the copy number of H1N1 M gene m RNA expression in lung tissue of mice with kidney yang deficiency;both single-drug and compound polysaccharide have significant proliferation effects on mouse spleen cells and macrophages.Meanwhile,MXF polysaccharides and asarum polysaccharides can reduce inflammation by regulating TNF-a,IL-6,IL-27,IFN-beta,IL-17,MCP-1 and other cytokines,which indicates that MXF polysaccharides and asarum polysaccharides can effectively regulate related signaling pathways and reduce inflammation caused by influenza virus infection.After comparison with PLS comprehensive efficacy,MXF polysaccharide is one of the important pharmacodynamic substances of MXF against influenza diseases. |
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