| ObjectiveBronchial asthma is a chronic airway immune-inflammatory disease characteriz-ed by airway remodeling,but its pathogenesis is not completely clear,which may be related to oxidative stress and immune-inflammatory factors.It has been found that many inflammatory cells and inflammatory factors participated in this process.There are various side effects associated with existing treatments,so many studies are now turning to herbal drugs.In order to explore the potential value and application prosp-ect of Phyllanthus amarus(PA)as prevention and treatment drugs of asthma,we studied the anti-inflammatory and antioxidant effects of PA on airway immune-inflammation in asthmatic rats and its possible mechanism.Methods(1)Preparation of PA extract:the aerial parts of PA were dried,powdered and extracted with methanol and dried by evaporation.The methanolic extract of PA was evaluated for phyllanthin and hypoph-yllanthin content by HPLC.(2)Experimental group:thirty-six SD rats were randomly divided into 6 groups,each group consist of 6 rats.Group I:normal control rats.Group II:AHR-control rats.Group III:MTL(10mg/kg)treated-rats.Group IV:PA low-dose(50mg/kg)treated rats.Group V:PA medium dose(100mg/kg)trea ted-rats.Group VI:PA high-dose(200mg/kg)treated-rats.(3)Establishment of asthmatic rat model:rats in Group ?-? were intraperitoneally injected with 1 mg OVA+300 mg aluminum hydroxide 0.66 ml on day 0,subcuta-neously injected with the same suspension on 7th day,and Group I was injected intraperitoneally and subcutaneously with the same amount of PBS.MTL(10 mg/kg)rats and PA(50,100 and 200 mg/kg)rats were administered by gavage from the 7th day to 35th day.Group II was given distilled water(1 Omg/kg)by gavage.On the 35-th day,except Group I,rats in other five groups were inhaled with 1%OVA(PBS)for fifteen min.(4)Detection of general condition,body weight,hemodynamic parameters,lung function and pathological changes of lung tissue:lung function was detected by noninvasive whole-body plethysmography within 24 hours after OVA challenge.The oxygen saturation(S02)was also measured at the meantime.Heart rate(HR),systolic blood pressure(SBP),diastolic pressure(DBP)and mean arterial pressure(MAP)were measured by carotid artery cannula.Airway inflammation,collagen deposition and mucus secretion were evaluated by HE,Masson and PAS staining respectively.The ultrastructural changes of lung were observed by transmission electron microscope.(5)Detection of the protein levels in blood and bronchoalveolar lavage fluid(BALF)of rats:Inflammatory cells were counted and classified by automatic cell counter and Field's staining.IgE and serum OVA specific immunoglobulin E(OVA-sIgE)were detected by enzyme-linked immunosorbent assay(ELISA).(6)Detection of oxidative nitrification stress related factors:the levels of malondialdehyde(MDA)and nitric oxide(NO)in the lung homogenate supernatant,and the levels of antioxidant markers such as superoxide dismutase(SOD)and glutathione(GSH)were detected.RT-PCR was used to detect the mRNA expression of nuclear factor E2 related factor 2(Nrf2),heme oxygenase-1(HO-1),inducible nitric oxide synthase(iNOS),transforming growth factor(TGF-?),tumor necrosis factor(TNF-?)and interleukin family(IL-1?,4,6).Results(1)Isolation and characterization of phyllanthin and hypophyllanthin from PA:the yield of a dry methanolic extract of PA was 42.5%(w/W).The HPLC analysis showed the presence of phyllanthin and hypophyllanthin at RT of 25.243 min and 26.832 min,respectively.The percent area for phyllanthin and hypophyllanthin were 68.14 and 31.95,respectively.(2)General changes of rats:OVA-induced rats of Group II were listless,less active,less eating and gasp after OVA-challenge.The symptoms of rats in MTL,PA treated-rats(Group ?-?)were significantly ameliorated.(3)Changes of rats' body weight and lung weight:in the Group II rats,the body weight decreased(P<0.05),and the relative lung weight increased(P<0.05).The body weight of Group ?-? rats increased(P<0.05),and the relative lung weight decreased(P<0.05).(4)Changes of hemodynamics parameters and oxygen saturation:SBP,DBP,MBP of AHR-control rats increased and HR decreased(P<0.05).But these hemodynamics parameters of Group ?-? rates were significantly improved(P<0.05),the improvement degree of MTL treated-rats was more obvious(P<0.05).SO2 of AHR-control rats was significantly lower than that of Group I(P<0.05).SO2 of Group ?-? increased(P<0.05),and that of PA treated-rats increased more significantly(P<0.05).(5)Changes of lung function:airway hyperresponsiveness(PIF,PEF,TV,EV,respiratory rate,Pehn)of AHR-control rats significantly increased(P<0.05).The lung function of Group ?,? and ? was significantly improved after MTL and PA treatment(P<0.05),and the effection of PA on lung function of Group? was more significant(P<0.05).(6)Changes of hematological indexes and inflammatory cell components in BALF:AHR-control rats' red blood cells(RBC)and related indexes decreased,and white blood cells(WBC)decreased(P<0.05).Group ?,? and V rats could basically restore the above changes to normal(P<0.05).The total number of inflammatory cells(neutrophils,lymphocytes,eosinophils and macrophages)in BALF of group ? were significantly higher than those in group I(P<0.05).The number of all kinds of cells in Group ?,?,? decreased significantly(P<0.05),and the change in Group ? was more significant(P<0.05).(7)Changes of total protein,albumin in serum,lung and BALF:total protein and albumin in serum,lung and BALF of AHR-control rats were significantly increased(P<0.05).Otherwise those of Group ?-? decreased(P<0.05),and the change of Group ? decreased more significantly(P<0.05).(8)Changes of serum total IgE and OVA-sIgE:the serum total IgE and OVA-sIgE in AHR-control rats were significantly increased after OVA-challenge(P<0.05).Group ?,V and ? were significantly lower(P<0.05),and Group ? was more significant(P<0.05).(9)Changes of oxidative nitrification stress level in lung tissue:oxidative nitrification stress level increased(MDA,NO)(P<0.05),antioxidant level decreased(SOD,GSH)(P<0.05).In Group ?,?,SOD and GSH increased(P<0.05),MDA and NO decreased(P<0.05),while only decrease of MDA and NO were found in Group V(P<0.05).Compared with the PA treatment,MTL treatment had more obvious effect on ameliorating oxidative nitrification stress level(P<0.05).(10)Changes of Nrf2,HO-1,iNOS and TGF-? mRNA expression in lung tissue:Nrf2 mRNA expression was down regulated(P<0.05),and HO-1,iNOS and TGF-? mRNA expression were up-regulated in AHR-control rats(P<0.05).The mRNA expression of the above factors in lung tissue of Group ?-?reversed(P<0.05).The expression of Nrf2 mRNA in Group ? was significantly higher than that of Group ?(P<0.05).(11)Changes of TNF-?,IL-1 ?,IL-4 and IL-6 mRNA expression in lung tissue:The TNF-?:IL-1 ?,IL-4 and IL-6 mRNA expression of AHR-control rats were signifi-cantly increased(P<0.05).The mRNA expression of the above factors in lung tissue of Group ?,? and ? reversed(P<0.05),and the downward trend of Group III was more obvious(P<0.05).(12)Pathological changes of lung:inflammatory changes(HE staining),interstitial fibrosis(Masson staining)and mucus hypersecretion(PAS staining)were observed after OVA-induced asthma in Group I(P<0.05).Pretreatment in Group ?,?,? could significantly improve the pathological changes of lung(P<0.05).Moderate edema and goblet cell proliferation were found in lung tissue of rats in Groups ? and ?.Conclusion:Phyllanthin and hypophyllanthin,the active components of PA,can ameliorate airway hyperresponsiveness,reduce the aggregation of inflammatory cells in BALF,and improve the pathological changes of lung tissue in OVA-induced AHR rats.The results showed that PA could reduce the oxidative nitrification stress(Nrf2,iNOS),and immune inflammatory reaction(HO-1,TNF-?,IL-1 ?,TGF-?1),decrease the levels of Th2 cytokines(IL-4,IL-6)and serum OVA-slgE,and significantly improve the changes of lung ultrastructure injury,thus improving airway remodeling.These results suggest that Phyllanthin and hypophyllanthin in PA may be a new method for the treatment of airway allergic diseases including asthma. |
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