Study On The Effect Material Basis Of Pinellia Ternata Decoction In The Treatment Of Airway Inflammation

Banxia is the tuber of Pinellia ternate（Thunb.）Breit.,belonging to the Araceae family,which has the effect of suppressing coughing,eliminating phlegm,reducing vomiting and dispersing nodules.It is used to cure mucous sputum and sputum due to cold,coughing,dizziness and headache due to sputum,vomiting,nausea,chest tightness,globus hysteriocus;and can be applied to cure swollen for external use.Some decotion with Banxia such as Erchen decoction,Banxiahoupo decoction and Chinese patent medicine such as Pinellia syrup,Pinellia cough syrup are widely used to treat airway inflammation such as bronchitis.Clinical analysis shows that Pinellia is frequently used in the treatment of diseases such as chest obstruction,phlegm dampness,and bronchitis,especially chronic bronchitis such as turbid phlegm blocking the lung and phlegm dampness accumulating lung.Pharmacological experiments show that Pinellia has anti-vomiting,suppressing coughing,eliminating phlegm,anti-inflammatory and anti-tumor effects.However,there is no relevant research on the effect components related to the quality of Pinellia and its processed products,and what kinds of components in Pinellia have these effects and the mechanism of Pinellia are still unclear.Therefore,an in vivtro airway inflammation model was established and a cell-level pharmacodynamic evaluation method,was used to study the mechanism of Pinellia ternata in treating airway inflammation,screens and isolates the effective parts of Pinellia and its processed products.The composition of the components is predicted based on network pharmacology,and part of the effect components of Pinellia decoction in the treatment of airway inflammation is carried out through cell experiments,and the material basis for the treatment of respiratory tract inflammation is preliminarily determined.The main research work is as follows:1.Therapeutic effect of Pinellia and Alum-processed Pinellia decoction on airway inflammation in mice modle.Mice were given LPS nasal drops（10 μg/mouse）on the 1st and 15th day,and be exposed to cigarettes smoke twice a day in a smoking cage during the rest of the day,4 cigarettes each time,5 days a week.4 weeks were needed to induce this modle.Pinellia decoction(3.0 g·kg^-1、1.5 g·kg^-1、0.75 g·kg^-1)was given by intragastric administration from the third week.After 14 days,mice were sacrificed,bronchoalveolar lavage fluid（BALF）and lung tissue were taken.Main bronchus was analysed by HE staining,the level of IL-6,IL-8 in BALF were detected via ELISA,the protein expression of MAPK,NF-κB signaling pathway were detected by Western blot.The results show that compared with the modle group,the high and medium doses of Pinellia decoction can significantly inhibited inflammatory cell infiltration,goblet cell hyperplasia and mucus secretion in main bronchial mucosa of mice;decrease the levels of IL-6 and IL-8,and also inhibit the phosphorylation level of p38 and IκB,but it has no effect on the phosphorylation level of ERK,JNK and p65.It indicates that Pinellia can treat airway inflammation by block the activation of p38-MAPK and NF-κB signal pathways.2.Comparative study on the therapeutic effects of Pinellia ternata and its processed product Alum-processed Pinellia decoction on miceAlum-processed Pinellia was processed according to the methods recorded in Chinese Pharmacopoeia 2020 Edition.The decoctions of Pinellia and Alum-processed Pinellia were prepared,and given to airway inflammation mice.The results showed that both raw Pinellia and Alum-processed Pinellia decoction can significantly reduce the expression of IL-8 and IL-6 in the BALF of airway inflamation mice,inhibit the phosphorylation level of p38 and IκB,and block the activation of p38-MAPK and NF-κB signaling pathways.There is no significant difference between raw Pinellia and Alum-processed Alum-processed Pinellia in reducing airway inflammation.It shows that the irritant toxicity of Alum-processed Pinellia is significantly reduced while the anti-inflammation effect still retains.This is consistent with the clinical use of Alum-processed Pinellia in the treatment of respiratory tract infectious diseases,indicating the scientific nature of the detoxification effect of Pinellia processing.3.Research on screening effective parts of Pinellia decoctionThe macromolecular components and small molecular components in the decoction of Pinellia ternata were separated by alcohol precipitation method.Airway inflammation mouse model and CSE induced 16-HBE cells were used to screen the effective parts of Pinellia decoction to cure airway inflammation.The expression of IL-6 and IL-8 in mouse BALF was detected by ELISA,and the expression of IL-6 and IL-8 mRNA in 16-HBE cells were detected by qPCR.Western blot was used to detect the protein expression of p-p38/p38 and pIκB/IκB in lung tissue of mouse and 16-HBE cells.The in vivo experiment showes that both the small molecular components（supernatant）and macromolecular components（precipitation）of Pinellia decoction(Equivalent to crude drug amount 3.0 g·kg^-1,1.5 g·kg^-1)can significantly reduce the levels of IL-6 and IL-8 in the BALF of airway inflammation mice.The in vitro experiment shows that Pinellia decoction(100 μg·mL^-1,50 μg·mL^-1)can significantly reduce the levels of IL-6 and IL-8 mRNA in 16-HBE cells induced by CSE,thus the effect of the small molecular components is slightly stronger than the macromolecular components.This effect is related to inhibiting the phosphorylation of p38 and IκB and blocking the MAPK and NF-κB signaling pathways.The small molecule components were separated by the system solvent extraction method,and the effect parts were screened by airway inflammation mouse model and CSE-induced 16-HBE cells.The in vivo experiment showes that the dichloromethane and ethyl acetate parts of Pinellia decoction(Equivalent to crude drug amount 3.0 g·kg^-1)could significantly reduce the expression levels of IL-6 and IL-8 in the BALF of airway inflammation mice,which related to the down-regulation the phosphorylation levels of p38 and IκB to block the activation of MAPK and NF-κB signaling pathways.But the n-butanol part and water part have no effect.The results of in vitro experiments show that ethyl acetate parts of Pinellia decoction(100 μg·mL^-1,50 μg·mL^-1)can significantly reduce the expression levels of IL-6 and IL-8 mRNA of 16-HBE cells induced by CSE.Therefore,the dichloromethane part and ethyl acetate part are the main effective parts of Pinellia decoction,while the n-butanol part and water part have no effect.And this is related to the down-regulation the phosphorylation levels of p38 and IκB to block the activation of MAPK and NF-κB signaling pathways.4.Analysis of the components in the dichloromethane and ethyl acetate parts of Pinellia ternata The HPLC-TOF-MS method was used to analyze the components of dichloromethane and ethyl acetate parts of Pinellia decoction ternata and Alum-processed Pinellia decoction,and the unknown components were inferred based on the fragmentation law.56 kinds of compounds were identified from the dichloromethane part of the raw Pinellia ternata,42 kinds of compounds were found in the dichloromethane part of the Alum-processed Pinellia ternata,70 kinds of compounds were identified from the ethyl acetate parts of the raw Pinellia,and 57 were found in the ethyl acetate part of Alum-processed Pinellia.In addition,there are 4 kinds of nitrogenous compounds with unknown structures in the dichloromethane and ethyl acetate parts of raw Pinellia and Alum-processed Pinellia.5.Comparison of the difference between alkaloid part and a non-alkaloid part in PinelliaIn order to further explore the pharmacodynamic material basis of Pinellia ternate Decoction in the treatment of respiratory tract inflammation,the non-alkaloid fraction（PTNAS）and alkaloid fraction（PTAS）were isolated by 732 cation-exchange resin,and the effects were compared with 16-HBE cell induced by CSE.The results showed that the non-alkaloid fraction could reduce the expression of IL-6 and IL-8 mRNA in 16-HBE cells induced by CSE.Combined with the components of dichloromethane and ethyl acetate and the adsorption characteristics of cation exchange resin,144 kinds of small peptide and 30 kinds of dipeptide were identified by Nano LC-MS and HPLC-TOF-MS/MS.The content determination results showed that the peptide content in PTNAS site reached 63.52%,indicating that the main components of Pinellia to treat respiratory inflammation may be peptides and organic acids.Summary1.We found that Pinellia decoction and Alum-processed Pinellia decoction can inhibit the phosphorylation level of p38 and IκB,down-regulate the contents of IL-6 and IL-8 in the BALF of airway inflammation mice.And there is no significant difference in the effects of Pinellia and its Alum-processed products,that is,alum process can still preserve its effect on the treatment of airway inflammation.2.The airway inflammation mice modle and the CSE-induced 16-HBE cell injury model were used to screen the effect parts of Pinellia decoction.The macromolecular and small molecular components separated from Pinellia decoction after alcohol precipitation can treat the airway inflammation mice,and the small molecular components（supernatant）has a stronger effect than macromolecular components（precipitation）.The dichloromethane part and the ethyl acetate part obtained have an effect in curing the airway inflammation.3.Through HPLC-TOF-MS technology,a variety of alkaloids,peptides,organic acids,amino acids and other components were identified from the effect site,and then it was separated to alkaloid parts and non-alkaloid parts（PTNAS）.In vitro experiments show that PTNAS can reduce CSE-induced 16-HBE cell IL-6,IL-8 mRNA expression.HPLC-TOF-MS combined nano-LC-MS/MS identified 144 kinds of small peptide and 30 kinds of dipeptide from PTNAS,and the BCA method determined peptide content in PTNAS is 63.52%.It suggests that the effect of Pinellia in treating respiratory inflammation may be related to the peptide components it contains.The above results laid the foundation for the treatment of respiratory inflammatory substances and the quality control of Pinellia.