The Prevention Effects Of Houttuynia Cordata Thunb.and Its Principle Compound 2-undecanone On B[a]P-induced Lung Carcinogenesis

Objective: In recent decades,lung cancer has been one of the fastest-growing cancers in the world for the incidence and mortality,and it has been increasing year by year.In the world,the incidence and mortality of lung cancer ranks first among male malignancies;among women,it ranks second only to breast cancer.Moreover,lung cancer is not easy to check,easy to transfer,rapid development and other characteristics,resulting in effective treatment of lung cancer has become a major global health problem.At present,clinical treatment of lung cancer is mainly focused on chemotherapy,radiation therapy,and surgical therapy.However,since lung cancer patients have been found mostly in the middle and late stages,the clinical treatment of lung cancer is now unsatisfactory,patients with lung cancer in developed countries are five-year survival rate is only 17.4%.Therefore,the effective prevention of the occurrence and development of lung cancer is an important to reduce the incidence and mortality of lung cancer.Air pollution and smoking are the main factors that cause the rising incidence of lung cancer.There are more than 73 carcinogens in tobacco,of which multi-chain aromatic compounds(such as benzopyrene)and nitrosamines are very strong.Carcinogenic activity,these carcinogens can lead to normal cell DNA damage and the formation of chronic inflammation microenvironment,and these factors will promote normal cell carcinogenesis.How to effectively prevent the occurrence and development of lung cancer has become a hot issue of public concern;whether it can prevent lung cancer by inhibiting DNA damage of normal lung cells and eradicating the micro-environment of chronic inflammation has become a research hotspot.Many studies have reported that Chinese herbal medicine has a unique and significant lung cancer prevention effect.Houttuynia cordata Thunb.is a traditional Chinese herbal medicine originating from the dried aerial part of the Chinese cabbage plant Amaranthaceae.It has been widely used as a medicine and food homologous medicine.Its main active pharmaceutical ingredient is methyl.N-nonanone and it is clinically used for the treatment of diseases such as phlegm,sore mass,acne hematochezia,and spleen and stomach heat,which are caused by heat and dampness.Modern pharmacological studies have shown that has antibacterial,anti-virus,improve the body’s immunity,diuresis and other effects,previous studies have shown that Houttuynia Cordata can also promote the apoptosis of A549 lung cancer cells.So,does Houttuynia have a role in preventing lung cancer? These key scientific issues have not yet been clarified and are worth studying.Based on the above research basis and clinical use,the study aimed to investigate the pharmacodynamic basis of Houttuynia cordata extract and its active monomer methyl n-nonyl ketone in preventing lung cancer,and further explored DNA damage inhibition and inflammatory mediators.Clear the mechanism of Houttuynia against lung cancer.In order to provide experimental basis for the prevention of lung cancer Houttuynia,and further promote the development and use of Houttuynia.Methods: 1.Inhibitory Effect of H.cordata and 2-undecanone with B[a]P on the BEAS-2B Cells H.cordata extract and 2-Undecanone were used to combination with B[a]P to the BEAS-2B cells for 48 h,The MTT used to assay for the cell proliferation.2.The effects of H.cordata and 2-Undecanone on the release of reactive oxygen species(ROS)in BEAS-2B cells were used to combination with B[a]P.B[a]P(5 μM)was applied to BEAS-2B cells for 0-48 h.Flow cytometry was used to detect the levels of ROS with time after administration,and the optimal administration time was selected.Antioxidant(NAC)was used as a positive control.Different concentrations of H.cordata and 2-Undecanone were combined with B[a]P(5 μM)in BEAS-2B cells for 48 h.The levels of ROS were detected by flow cytometry.3.The effects of H.cordata and 2-Undecanone on DNA damage and inflammatory of BEAS-2B cells were combined with B[a]P.Antioxidant(NAC)was used as a positive control.H.cordata and 2-Undecanone were co-treated with B[a]P(5 μM)in BEAS-2B cells for 48 h.The expression of DNA damage marker p-h2 a.x was detected by Western blot and confocal microscopy.B[a]P(5 μM)was applied to BEAS-2B cells for 0-24 h.The secretion of IL-1β,the intracellular inflammatory marker,was detected by Western blot,and the optimal administration time was selected.Antioxidant(NAC)was used as a positive control.Different concentration of H.cordata and 2-Undecanone were used together with B[a]P(5μM)in BEAS-2B cells for 24 h.Western blot was used to detect the secretion of inflammatory marker-IL-1β in the cells.4.The regulation effect of H.cordata and 2-Undecanone on antioxidant protein H.cordata and 2-Undecanone were used to combination with B[a]P for BEAS-2B cells for 12 h.Western blot was used to detect the nuclear translocation of Nrf2 in cells and the expression of antioxidation.protein HO-1 and NQO-1.5.The preventive effect of H.cordata and 2-Undecanone on B[a]P-induced lung cancer in mice B[a]P-induced lung cancer model of A/J mice was established.H.cordata and 2-Undecanone were administered for 36 weeks to examine the preventive effect of drugs on lung cancer.During the administration,body weight was measured once a week and the morphology of the animals was observed.At the end of the experiment,the number of pulmonary nodules was calculated.Western blot,IHC,IF and ELISA techniques were used to detect the expression of Nrf 2,DNA damage markers,and inflammatory markers.Results: 1.H.cordata and 2-Undecanone can significantly reverse the inhibitory effect of B[a]P on the proliferation of BEAS-2Bcells,and there is no obvious inhibitory effect on the proliferation of BEAS-2B cells when administered alone.MTT assay showed that B[a]P significantly inhibited the proliferation of BEAS-2B cells 48 h after administration,and H.cordata and 2-Undecanone were no significant inhibitory effect on the value of BEAS-2B cells in the concentration range.H.cordata and 2-Undecanone combined with B[a]P on BEAS-2B cells for 48 h,And H.cordata and 2-Undecanone significantly reverse B[a]P inhibition of cell proliferation.2.H.cordata and 2-Undecanone significantly reduce the increase of ROS in BEAS-2B cells induced by B[a]P.The results of flow cytometry showed that within 48 hours after administration,B[a]P induced intracellular ROS gradually increased;H.cordata and 2-Undecanone with B[a]P on BEAS-2B cells for 48 h,H.cordata and 2-Undecanone significantly reduce the ROS produced in a concentration-dependent manner.The highest concentration effect was close to the positive control.3.H.cordata and 2-Undecanone reverse B[a]P-induced DNA damage and reduce the release of inflammatory mediators of BEAS-2B cells.The extract of H.cordata and 2-Undecanone were used to combination with B[a]P for 48 hours.The results of Western blot and Confocal microscopy showed that the expression of P-H2 A.X,a marker of DNA damage in cells,was reduced and showed concentration-dependent.Dependence,the highest concentration effect is close to the positive control.Within 48 h after administration of B[a]P,the secretion of IL-1β,an inflammation marker,first increased and then decreased,reaching its peak at 24 h.H.cordata and 2-Undecanone were used in conjunction with B[a]P in BEAS-2B cells for 24 h.Western blot results showed that the secretion of IL-1β,an inflammation marker,was decreased in a concentration-dependent manner.The highest concentration effect was close to positive group.4.H.cordata and 2-Undecanone induce the expression of anti-oxidation protein After H.cordata and 2-Undecanone acted on BEAS-2B cells for 12 h,Western blot results showed that the nuclear translocation of Nrf 2 increased,and the expression of antioxidant proteins HO-1 and NQO-1 increased;H.cordata and 2-Undecanone were used to combination with B[a]P for BEAS-2B cells for 12 h.Western blot results showed that H.cordata and 2-Undecanone reverse the B[a]P-induced reduction of Nrf 2 nuclear entry and antioxidant activity protein HO-1 and NQO-1 expression decreased.5.Mechanism of action of H.cordata and 2-Undecanone in preventing lung cancer in vivo.In the B[a]P-induced lung cancer model of A/J mice,H.cordata and 2-Undecanone significantly inhibit the occurrence and development of tumors.During the gavage for 38 weeks,there were no significant fluctuation in body weight in all groups,and there were no significant difference in organ index.The results of Western blot,IHC,IF and ELISA showed that the expression of P-H2 A.X,a marker of DNA damage,was significantly decreased in the group treated with drug,and the expression of IL-1β,an inflammation marker,was also significantly reduced.Conclusion: In vitro,H.cordata and 2-undecanone promote the nuclear translocation of Nrf2,remove ROS to inhibit DNA damage and the release of inflammatory corpuscle,to protect the damage of normal cells.In vivo,H.cordata and 2-undecanone inhibit the occurrence and development of lung cancer by inhibited DNA damage and release of inflammatory mediators.The results of this study clearly clarified the effect and mechanism of H.cordata on lung cancer prevention.It can provide experimental basis and data support for the clinical application of H.cordata to prevent lung cancer.