Research On The Quality Standard And The Effect On Inhibiting PQ-induced Pulmonary Fibrosis In Mice Of Lotus Plumule Extracts

Lotus plumule is the green radicle in the middle of Nelumbo nucifera Gaertn.ripe seeds,main active components are alkaloids,including liensinine,isoliensinine,neferine and so on.Modern pharmacological studies have found that lotus plumule has the functions of anti-oxidant,anti-inflammatory,anti-arrhythmic,anti-cancer,and lowering blood sugar.In this paper,the contents of three alkaloids in lotus plumule extracts were determined simultaneously by HPLC,and the effects and mechanism of lotus plumule extracts on inhibiting PQ-induced pulmonary fibrosis in mice was preliminarily studied.Part One Study on the quality standard of the Lotus Plumule extractsObjective:The aim of this part was to establish the quality standard of the LP extracts and determine the contents of the main components.Methods:The chromatographic separation was performed on a Gemini-NX5U C18 column with elution of acetonitrile–methanol-water-triethylamine(40:20:40:0.12).The flow rate was 1.0 mL·min~–11 and the column temperature was 30?.Results:All compounds showed satisfactory linearity(r>0.9995)within the ranges.The method validation showed satisfactory precision,accuracy,stability,and the overall recoveries of 99.7-101.1%for the three compounds.Conclusions:The quality standard is sensitive,accurate,repeatable and specific,which could ensure the quality control of LP extracts.Part Two Research on the effect of lotus plumule extracts on inhibiting PQ-induced pulmonary fibrosis in miceObjective:The aim of this study was to investigate the effects of Lotus Plumule Extract on inhibiting PQ-induced pulmonary fibrosis in mice and preliminarily explore its possible mechanism.Methods:1 Seventy male Kunming mice of 18±2 were randomly divided into 7groups including normal control(NS)group,PQ model(PQ)group,LP low-dose(LP-L)group,LP middle-dose(LP-M)group,LP high-dose(LP-H)group,vitamin C(Vc)group and dexamethasone(DEX)group.The mode of pulmonary fibrosis was induced by a single intraperitoneal injection with paraquat(PQ).24 hours after injection with PQ,the medicine-treated groups were given corresponding medicine,while the NS and PQ groups were instead treated with 0.9%saline.After 21 days,all the mice of groups were sacrificed.2 The mice of groups were observed changes in general condition and weight during experimentation period.The mice were sacrificed at 21 day after modeling and the lung tissues were collected and observed.The degree ofinflammationandpulmonaryfibrosiswereevaluatedby haematoxylin-eosin(HE)and Masson trichrome(Masson)staining.3 The content of malondialdehyde(MDA)and superoxide dismutase(SOD)were determined in order to evaluate the effects of LP on oxidative stress of pulmonary fibrosis mice by enzyme-linked immunosorbent assay(ELISA).4 The expression level of Col-I in the lung tissue of mice was tested by immunohistochemical staining and was analyzed.5 Total RNA was extracted from the lung tissue of mice.The expression levels of IL-6,IL-17 mRNA were detected by RT-PCR.6 The content of transforming growth factor beta-1(TGF-?1)as an important signal factor in the process of pulmonary fibrosis was determined by ELISA.The content of hydroxyproline(HYP)was also determined by ELISA.Results:1.The appetite,mental status and the fur of mice were normal in the NS group.While in the PQ group,the appetite,mental status and the fur of mice became worse,and the body weight decreased.Subsequently,the body weight of mice increased gradually,but still lower than that of NS group(P<0.01).The body weight of LP-L?LP-M?LP-H groups were significantly improved compared with PQ group at 3 weeks(P<0.05,P<0.01).2.According to morphologic survey,the texture and elasticity of the lung in the NS group were in normal state and that of PQ group became worse.The damage degree of LP groups decreased compared with that of PQ group.3.The result of HE and Masson staining showed that there are obvious increase of inflammation and deposition of collagen in PQ group compared with that of NS group(P<0.01).The degree of inflammation and fibrosis in LP-M,LP-H groups decreased compared with that of the PQ group(P<0.05,P<0.01).4.The content of MDA in serum of the PQ group was significantly higher than that of the NS group,while the content of SOD in serum of the PQ group was obviously lower than that of the NS group.After treated with LP,the level of MDA and SOD achieved different degree decrease and improvements,the effect of LP-H was more significant than that of the PQ group(P<0.01).5.According to the immunohistochemistry analysis of Col-I,the expression of Col-I increased significantly in the PQ group compared with the NS group(P<0.01).Obvious reduction of expression of col-1 in the LP-H,LP-M and DEX group compared with the PQ group,while it was not significant in LP-L group.6.The mRNA expression of IL-6,IL-17 was detected by RT-PCR.No significant change of IL-6,IL-17 in the NS group.IL-6,IL-17 mRNA in PQ group was significantly higher than that in the NS group.The mRNA expression of IL-6,IL-17 decreased significantly in DEX,LP-M,LP-H group compared with the PQ group.7.The level of TGF-?1 and HYP was detected by ELISA assay,which was significant lower in the DEX group and LP-H group compared with the PQ group.Lower expression of TGF-?1 and HYP also found in LP-L group,but not significant.Conclusions:The mechanism of therapy of LP might reduce pulmonary inflammation,and inhibit oxidative stress to improve anti-pulmonary fibrosis ability,and decrease the expression of TGF-?1 to down-regulate the collagen deposition to prevent pulmonary fibrosis.