| ObjectiveIn order to explore the anti-lung cancer activity of Houttuynia cordata volatile oil and sodium new houttuyfonate in vivo and in vitro,and the related mechanisms of the two drugs inducing apoptosis in A549 cells.At the same time,in order to solve the disadvantages of volatile oil and low bioavailability when using Houttuynia cordata volatile oil,the preparation of encapsulation rate meets the requirements,and the long-acting slow-release polyethylene glycol modified Houttuynia cordata volatile oil liposome.MethodIn this study,the MTT method was first used to detect the proliferation inhibition rate of Houttuynia cordata volatile oil and sodium new houttuyfonate on A549 cells.FITC-AnnerxinV/PI double staining method was used to detect the apoptosis of sodium new houttuyfonate,and PI single staining was used to detect the cell cycle distribution.The acute toxicity test investigated the toxicity of Houttuynia cordata volatile oil and sodium new houttuyfonate,determined the concentration of the drug,and carried out subcutaneous tumor-bearing experiments in nude mice.The Houttuynia cordata volatile oil and sodium new houttuyfonate sodium were used to intervene in A549 cells,GAPDH was used as the internal reference gene,and real-time quantitative PCR was used to detect the expression of related genes and Western blotting to detect the expression of related proteins.Preparation of polyethylene glycol modified Houttuynia cordata volatile oil liposomes by film dispersion method,with egg yolk lecithin/volatile oil,egg yolk lecithin/cholesterol and PEG2000-DSPE mass fraction as the main factors,encapsulation rate evaluation index,Box-Behnken response the surface method optimizes the preparation process.The appearance,particle size,potential,and PDI size of the preparation were investigated,and the stability factor test was conducted.Gas chromatography method was established to detect the content of Houttuynia cordata volatile oil in rat plasma,and a methodological investigation was conducted.Blood was collected from the orbit of the rat,centrifuged to collect drug-containing plasma,and gas chromatography was used to detect drug concentration.Draw the drug-time curve and use DAS 3.0 software to get the relevant pharmacokinetic parameters.ResultMTT test results showed that Houttuynia cordata volatile oil and sodium new houttuyfonate all inhibited the proliferation of A549 cells,and had obvious concentration and time dependence.Sodium new houttuyfonate inhibits the proliferation of A549 cells mainly in the early stage of apoptosis.With the increase in drug concentration,the late stage apoptosis rate slightly increased,and the total apoptosis rate showed an upward trend,which was statistically significant between each group(P<0.05).Compared with the negative control group,the number of cells in G0/G1 phase increased,the number of cells in S phase decreased,and the number of cells in G2/M phase remained basically unchanged(P<0.05).In the acute toxicity test,the LD50 values of Houttuynia cordata volatile oil and sodium new houttuyfonate sodium were 1904 mg/kg and 197 mg/kg,respectively.The dosage of Houttuynia cordata volatile oil and sodium new houttuyfonate in tumor-bearing mice was 250 mg/kg,200 mg/kg,150 mg/kg;40 mg/kg,30 mg/kg,20 mg/kg.The results showed that the tumor inhibition rate of cisplatin was 61.97%,Houttuynia cordata volatile oil was up to 65.41%,and sodium new houttuyfonate sodium was up to 62.62%,both of which were concentration-dependent.Compared with the control group,the Houttuynia cordata volatile oil group and the sodium new houttuyfonate sodium group both had significant differences in tumor-inhibiting rates(P<0.05).Pathological section results showed that the Houttuynia cordata volatile oil and sodium new houttuyfonate had a certain inhibition on the transplanted tumors growth in A549 mice,and there was a certain dose dependence,and the toxic and side effects on other organs were not obvious.In this paper,after intervening with Houttuynia volatile oil,GAPDH was used as an internal reference gene to determine the six genes in the RAF/MEK/ERK-Bcl-2 signaling pathway:RAF-1,MEK-1,MEK-2,ERK-1,ERK-2 and Bcl-2,expression levels were tested.The results showed that the Houttuynia cordata volatile oil had an effect on the expression of four genes,RAF-1,MEK-1,ERK-1 and Bcl-2,and had no effect on MEK-2 and ERK-2.With the increase of the administration concentration,the expression levels of the four genes showed a downward trend to varying degrees,and there was a significant difference from the negative control group(P<0.05).Western blotting test results show that Houttuynia cordata volatile oil had no significant effect on the protein expression of RAF-1,MEK1/2 and ERK1/2,but their phosphorylated protein expression level was significantly reduced.The relative protein expression of Bcl-2 showed a downward trend.As the concentration increased,the expression levels of P-RAF1,P-MEK1/2,P-ERK1/2,and Bcl-2 gradually decreased,and there was a significant difference from the negative control group(P<0.05).Using the new houttuyfonate sodium intervention,GAPDH was used as the internal reference gene to detect changes in the expression levels of TNF-? and NF-?B in related pathways.The results showed that the expression of TNF-? and NF-?B genes by new houttuyfonate sodium influential.With the increase of the administration concentration,the expression levels of the two genes decreased in different degrees,and there was a significant difference with the negative control group(P<0.05).Western blotting test results show that the relative protein expression of IKKa was not significantly affected,but the corresponding phosphorylated protein expression level was significantly reduced.The NF-?B p65 protein and its phosphorylation level also showed a downward trend.As the concentration increased,the expression levels of P-IKKa,P-NF-?B p65 and NF-?B p65 gradually decreased,and there was a significant difference from the negative control group(P<0.05).The best prescription for preparing polyethylene glycol modified Houttuynia cordata volatile oil liposome:egg yolk lecithin/volatile oil is 4.6:1,egg yolk lecithin/cholesterol is 3,6:1,and the mass fraction of PEG2000-DSPE is 2%.The encapsulation efficiency and drug loading of the prepared liposomes were 80.42 ±0.12%and 8.35 ± 0.01%,respectively.The average particle size is 114.43 ± 1.39 nm,PDI is 0.27± 0.01,Zeta potential is-25.8 ± 0.25 mV,and the release experiment shows that the liposome has a certain sustained release effect.Gas chromatographic method was used to detect the content of Houttuynia cordata volatile oil in rat plasma.The method has good precision,small error,and sTab.and reliable results.The standard curve is Y=0.061X+0.0672,R2?0.9992,and the linear relationship is good in the linear range of 0.15 ?g/mL?149.70?g/mL.After the Houttuynia cordata volatile oil was prepared into polyethylene glycol modified liposome,its half-life T1/2 was extended by 1.69 times,the elimination rate CL/F was decreased by 1.56 times,and the area under the curve AUC increased by 1.89 times.ConclusionHouttuynia cordata volatile oil and sodium new houttuyfonate all inhibited A549 cells in a time and concentration dependent manner.Both Houttuynia cordata volatile oil and sodium new houttuyfonate can inhibit the growth of tumor tissue,and the maximum inhibition rates can reach 65.41%and 62.62%,respectively.Houttuynia cordata volatile oil may induce A549 cell apoptosis by inhibiting the expression of RAF/MEK/ERK pathway related genes and protein phosphorylation levels,thereby down-regulating the expression of Bcl-2 and inducing apoptosis in A549 cells.Sodium new houttuyfonate may induce A549 cell apoptosis by inhibiting the expression of NF-?B pathway-related genes and protein phosphorylation levels.The prepared polyethylene glycol modified Houttuynia cordata volatile oil liposome encapsulation rate meets the requirements,can extend the half-life,improve the bioavailability and achieve the effect of long-term slow release. |
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