Based On Microfluidic Chip Technology, The Preliminary Study On The Distribution Rule And Mechanism Of Nepeta Flavonoids Against Lung Cancer

Objective:The research was supported by national “The build of integrated microfluidic chip to screen the compatibility of multi-component of traditional Chinese medicine compounds and screening of new drug” “twelfth Five-Year Plan” “The drug discovery initiative”major science and technology projects(Project Number: 2013ZX09507005-005)and General project of education department of Liaoning province(Project Number: L201605).The purpose was to explore the intervention effect of total flavonoids from Schizonepeta tenuifolia(TFS)against the human lung cancer cell based on the microfluidic chip technology particularly the Non-small cell lung cancer(NSCLC),investigate its effective composition and compatibility principle,and making a deep research about the preliminary molecular mechanism.In a word,all these works were carried out to provide a mode of quality control which was guided by the pharmacology pharmacodynamics and reference data for the development of new drugs against lung cancer.Method:1.Using three major technical means to build a chip for pharmacodynamic screening which including soft lithography,injection molding and plasma bonding,and the applicability of the chip was investigated through some related fluorescence reagents.2.Apoptosis and necrosis rate(%)was regarded as the index of evaluation based on microfluidic chip technology,fluorescence staining reagents of Hoechst 33342 / Propidium iodide(PI)were used to carry out the intervention effect of TFS against human lung cancer cell line A549 between different groups and different medicinal parts in vitro experiment.3.The Fingerprint of TFS and content detection was established by the method of High Performance Liquid Chromatography(HPLC).The analysis software of grey relational degree was used to set up a spectrum effect relationship.High Performance Liquid Chromatography-Q-time-of-flight mass(HPLC-Q-TOF-MS)was used to analysis and confirm the exact chemical compositions about the pharmacodynamic components accurately.4.We search the pharmacodynamics research in vitro with the verified pharmacodynamic components,and apoptosis and necrosis rate(%)was regarded as the index of evaluation.Under this condition,the optimal proportion against the lung cancer cells between the pharmacodynamic components was screened with the help of uniform design.5.Annexin V-FITC/PI double staining and PI staining method were used to detect the cell cycle and cell apoptosis through Flow Cytometry after medicine stimulation.6.The technology of Polymerase Chain Reaction(PCR)and Western Blot were adopted to detect Micro RNA and related expression of genes in human lung cancer A549 cell after TFS and pharmacodynamic components stimulation.Aiming to explain a part of mechanism on lung cancer from the level of genes clearly.Results:1.A double-structure chip was designed and manufactured into a product with polydimethylsiloxane(PDMS)in this experiment.Sixteen air valves and eight liquid valves were integrated on the first layer.The former was named as A,B,C,D,E,F and G,while the latter was named as a,b,c,d,e,f,g and h.At the same time,aa1 were regarded as a pair for use among them.The second layer contains 4*4 column elliptical structures and appropriate fluid channel structure which was called cell culture chamber,the chip shared the same outlet for waste accordingly.Test results showed that the chip which integrated with microvalves have a good quality.On the one hand,the valves not only can controlled isolation within 72 hours,but also can open successfully after 72 hours.In a word,it has a sensitive rebounding effect.On the other hand,the lung cancer cell of A549 which cultured in chip showed a stable growth condition without obvious difference compared with cells under routine cultivation.The survival ratio was beyond 98%.All these results displayed that the chip can be used for later experiment research.2.The results which about different action of TFS based on the microfluidic chip technology against human lung cancer cell showed that TFS has an inhibitory effect on lung cancer cell,and different origin have a certain difference.Anhui was the best,apoptosis and necrosis rate achieved(67.40 ± 3.56)%,Guangdong was the worst,apoptosis and necrosis rate only achieved(10.91 ± 1.26)%.The pesticide effects of other five origins are close to each other.Meanwhile,the strength of different medicinal parts was ordered as follow: leaf > root > stem > flower.All these results were all under the same action time and concentration.3.We established the Fingerprint of the TFS,and analyzed the similarity through evaluation software.The results showed that the similarity was variable between difference habits.All in all,the results can be divided into 3 types,the similarity of the first type was all beyond 0.85 including S1(Anhui),S2(Gansu),S5(Henan-shangqiu),S7(Hubei),S8(Sichuan)and S9(Yunnan).S6(Henan-bozhou)was the second type.The similarity was more than 0.85.The third type was composed of S3(Guangdong)and S4(Hebei),the similarity were all around 0.36.Furthermore,the relationship analysis between the spectrum and efficacy indicated that twenty common peaks have a high correlation to anti-lung cancer effect.The sequence of correlations was ordered from strong to weak as 19>6>10>12>20>16>11>18.In view of the above analysis results,we analyzed and identified the related pharmacodynamic components including quercitrin,hesperidin,apigenin,diosmetin,luteolin,icynaroside through HPLC-Q-TOF-MS.4.The results about the content detection which was tested by HPLC showed that different origin have some differences.Detailed results were presented as follow.All the content was varied from 7.58 mg·g-1 to 17.91 mg·g-1,and the Anhui was the best among other origins.Subsequently,a clustering analysis also displayed that the nine habitats were divided into three parts.The first class comes from Anhui province,the second class was formed by Henan-shangqiu and Guangdong.At last,the third category including Henan-bozhou,Hebei,Yunnan,Sichuan and Gansu.5.Six components performed some differences under the same concentration condition.For example,the results of the effect displayed as follow: icynaroside > quercitrin> hesperidin > apigenin > diosmetin > luteolin.While the results of the uniform design showed that all the compatibility groups can induce lung cancer cell apoptosis in a certain degree.The apoptosis and necrosis rate varied from(27.68 ± 1.67)% to(81.00 ± 5.15)% among these groups.Group 3 was the best.At last,we screened the best compatibility with the help of CSZ software as follow.Hesperidin: luteoloside: quercitrin: luteolin: apigenin:diosmetin = 3.06: 2.90: 2.04: 4.17: 0.12: 2.75.6.All the results displayed that TFS and six pharmacodynamic components have the ability to induce lung cancer cell apoptosis according to the Flow cytometry.Apoptosis and necrosis rate of the compatibility group 3 can reach to(67.9 ± 1.2)%,and high-dose group of TFS was the second which can achieved(52.5 ± 0.7)%.The other groups were ordered by efficiency from strong to weak as follow.Icynaroside > quercitrin > hesperidin >middle-dose group of TFS > apigenin > diosmetin > luteolin > low-dose group of TFS.Besides,those components also can inhibit lung cancer cell division effectively.They can increase the proportion of phase S and postpone the development of the lung cancer cells through inhibiting the transformation from phase G0/G1 to phase S.7.The results of PCR showed that the expression of some key molecules in PI3K-AKT signaling pathway was intervened,such as micro RNA 126(mi R-126)and PTEN up-regulated,while the expression of VEGF and PI3 K down-regulated after the TFS stimulation on lung cancer cells A549.At the same time,the six components and the best compatibility group also can regulate the expression of PTEN,VEGF and PI3 K at the same trend.8.The result of Western blot showed that the expression of some key molecules in PI3K-AKT signaling pathway was intervened,such as VEGF,AKT,Bcl-2 and Cyclin B1 on lung cancer cells A549 was down-regulate,up-regulate the protein expression of PTEN and PI3 K after TFS stimulation.While six components and the best compatibility group can decreased the protein expression of AKT and Cyclin B1.Luteolin can decrease the expression of PI3 K,apigenin,while diosmetin and hesperidin can increase the expression.All the groups can up-regulate the expression of PTEN,down-regulate the expression of the Bcl-2 except the best compatibility group and hesperidin.Conclusion:1.A microfluidic chip for screening of anti-lung cancer cells was constructed in this experiment which has many advantages including flexible operation,easy integration,high success rate,stable result and low production cost.It can meet the current experimental requirements.So far,it can be successfully applied to pharmacodynamics research including different origins and different medicinal parts.2.The results of pharmacodynamic testing which based on microfluidic chip showed that different areas and medicinal parts have different effects on lung cancer cell in vitro experiments because of different climate and environment.Anhui is one of the main origin of Schizonepeta tenuisfolia,and its effect was the best among others.The results of different medicinal parts showed that the root also had the same effect on inducing lung cancer cells apoptosis.So it can expand the roots as medicine.In summary,this research can provide a theoretical basis not only for reasonable application but also development of drug therapy for lung cancer.3.In this research,we obtained the Fingerprint of TFS and pharmacodynamic components with the technology of HPLC,software of grey relational degree and HPLC-Q-TOF-MS.The content of multi index components including six identified chemical compositions was also detected on this basis.The method was stable and feasible,which can provide a reference for the quality control for Schizonepeta.4.The research had initially completed the compatibility study which the results showed that those six pharmacodynamic components have different effect on lung cancer A549,and explained the efficacy of a mixture is not the simple ingredients superposition.The results showed that the compatibility group was better than the same dose of TFS.Above experimental results,the optimal proportion of six components which obtained from the part of uniform design laid the foundation for further research deeply.5.All those groups including TFS,six pharmacodynamic components and the best compatibility group have an effect on inducing cell apoptosis and inhabiting cell division within a certain concentration range.However,the target was different.The TFS can regulate Sphase significantly,the monomer composition can regulate G0/G1 phase,S phase,and G2/M phase respectively.These results suggested that the synergistic interaction of monomer components could interfering the S phase division,and antagonism G0/G1 phase and G2/M phase.These results fatherly verify the multiple-target concept and synergistic effect of Traditional Chinese Medicine(TCM)which have a variety of chemical components clearly.6.Schizonepeta has a complex chemical composition.On the one hand,it can induce cancer cells apoptosis through interfering PI3K/AKT signaling pathway,in this research,we can easily see that different groups up-regulated the expression of the PTEN,down-regulated the expression of the PI3 K and VEGF in some degrees.On the other hand,it has been proved that VEGF is a target of mi R-126.Mi R-126 plays an important role in both PI3K/AKT and VEGF signaling pathways.In this research,TFS has participated in regulation of some metabolic network in body through regulating the expression of some related proteins.All in all,initially clarified the anti-cancer mechanism of TFS.7.From the view of molecular biology,we can see that the TFS can regulate the PI3K/AKT signaling pathway and regulate the expression of related proteins.We should note that these proteins were not only controlled by their corresponding genes,such as Cyclin B1 and Bcl-2.All these performed obviously in each groups,while only the Bcl-2 have no significance in compatibility group compare with control group.Taking all these results into consideration,we can draw a conclusion that the anti-lung cancer effect of TFS was owing to the various components exit in Schizonepeta,and they cooperate with each other.Rather than a simply summarize of single mechanism.