Study On The Synergistic Effect Of Shenqi Fuzheng Injection Combined With Cisplatin In The Treatment Of Lung Cance

Lung cancer is the most common and fatal lung malignant tumor in the world,which has the highest mortality rate among malignant tumors in recent years.Because of the undeveloped nerves in the lungs,lung cancer often has no symptoms in the early and middle stages.As a result,about 86%of the patients have reached the advanced stage of lung cancer at the time of diagnosis.At that time,most patients have lost the chance of surgery,so they were mainly treated by radiotherapy,chemotherapy and molecular targets therapy.According to statistics,the 5-year survival rate of patients with non-small cell lung cancer(NSCLC)is only 10%to 15%,and that of small cell lung cancer(SCLC)is only 5%.Moreover,due to the liver and kidney toxicity,ototoxicity and decreased immunity caused by radiotherapy and chemotherapy,the use of drugs is restricted and the quality of life of patients is low.Synergistic treatment of Chinese and Western medicine has obvious effects on lung cancer patients in improving symptoms,improving quality of life and prolonging survival.Cisplatin(DDP)is a periodic non-specific anti-tumor drug,which is also one of the most commonly used broad-spectrum anticancer drugs in clinic and is used as a first-line chemotherapeutic drug for various tumors.However,with its anti-tumour effects,DDP is easy to induce toxic and side effects on the bone marrow,digestive system,kidney and inner ear of the body,and the long-term or high-dose use of DDP is easy to induce drug resistance,which will reduce its efficacy.SQFZ Injection is a pure traditional Chinese medicine intravenous infusion for the treatment of "fuzhengbuqi" and assisting anti-tumour,which belongs the second class of new Chinese medicine.Its main raw materials are Codonopsis pilosulae and Astragalus membranaceus,which contains chemical components such as polysaccharides,saponins,lignans and flavonoids.Codonopsis saponins can enhance immune cell function,improve appetite,reduce fatigue,and improve autonomic function to a certain extent.Codonopsis pilosula polysaccharide has the effect of strengthening humoral immunity,hindering the development of tumor cells,and resisting tumor cell metastasis.The Astragalus polysaccharide and Astragalus saponin in Astragalus membranaceus can enhance the activity of immune cells.It has been reported that SQFZ was mainly used in the clinical treatment of lung cancer and gastric cancer by improving the immunity of patientsthe quality of life.There are more and more applications of combined use of Chinese and Western medicine in the treatment of diseases with synergistic effects.However,there are few reports on the synergistic effect and synergistic mechanism of DDP and SQFZ.Based on this,the network pharmacological analysis was firstly used in this study to construct network targets related to DDP,SQFZ and NSCLC molecules.After further literature studies on these targets,it was proposed to select them as indicators for subsequent animal and cell experiments.Then,Then cisplatin,shenqi fuzheng single drug and their combination were applied to A549/LLC cells to explore whether their combination use could effectively enhance the anti-lung cancer effect.In addition,relevant targets were selected based on network pharmacology results,potential mechanism of synergy between the two was explored using Western Blot,and the pharmacodynamics was finally verified the through animal experiments.The specific research contents are as follows:1 Construction of anti-lung cancer molecular network of SQFZ and DDPFirstly,DDP and the main components of SQFZ:5-hydroxymethylfurfural,Syringin,Astragaloside IV,Astragaloside I,Astragaloside II,Astragaloside III,Formononetin,Calycosin,Vernine,Coniferin,Vanillic acid and Lobetyolin were set as Key words.Then,the drug-related genes were searched using the Genecards(http://www.genecards.org/)database.In the background of "homo sapiens",the related gene targets with high reliability(score>3.0)in the Genecards database were extracted,and de-weighted and their names were unified.Among them,451 targets were obtained from cisplatin and 255 targets were obtained from SQFZ.Non-small cell lung cancer was submitted to the DisGeNET database,and the human gene was used as the background to extract relevant gene targets with high reliability(score>2.0)in the database,.Those targets were deweighted and their names were unified.A total of 3,940 genes related to non-small cell lung cancer were obtained.The gene targets of DDP,SQFZ and NSCLC were mapped using Cytoscape 3.7.0 software to obtain a"drug/component-target-disease" network map.The identified drug/ingredient-target-disease network was imported into the ClueGO of Cytoscape software for KEGG pathway enrichment analysis.Then,the obtained KEGG pathway results were imported into the KEGG(http://www.kegg.jp)website for access query.Further verification by literature confirmed that CASP3,CASP9,BAX,BCL-2,JAK2,STAT3,P50,P65 and VEGF were used as indicators of in vitro and in vivo experiments for the combination use of SQFZ and DDP against lung cancer.2 Effect of SQFZ injection combined with DDP on proliferation,morphology,apoptosis of A549/LLC cells and its mechanismAt first,different concentrations of SQFZ(0.01ml/L,0.1ml/L and 1ml/L)and cisplatin(0.001 μg/ml,0.01μg/ml,0.1μg/ml,1μg/ml and 5μg/ml)were applied alone or in combination on A549 and LLC cells.Then,the growth inhibition effect of the drugs on lung cancer cells was evaluated by MTT colorimetry.Results showed that the single use of SQFZ at different concentrations did not significantly inhibit the growth of A549 and LLC cells;cisplatin alone inhibited the growth of lung cancer cells.The higher the concentration of cisplatin was,the more significant the inhibitory effect was,showing a concentration correlation inhibitory effect;compared with groups of cisplatin alone at different concentrations,the combination use of SQFZ and DDP could increase the cell inhibition rate.According to the CDI,It was found that 0.01 ml/L SQFZ and DDP exerted the best synergistic effect.After treated by DDP and SQFZ for 24h,A549 cells were stained with DIL and DAPI.DIL stained the cell membrane red;DAPI stained the cells with blue.Then,the cells were observed under laser confocal microscopy.Compared with the blank group,the cell nucleus and cell membranes of SQFZ groups showed no significant changes.Although the nucleus of cisplatin group was intact,the cell membranes were damaged and deformed,and the cell membrane damage was more serious after the combination use with SQFZ,indicating that the combination of the two drugs produced a synergistic effect.After A549 cells were treated with cisplatin and Shenqi Fuzheng separately and in combination for 24h,Annexin V-FITC and PI double-stained cells were detected by flow cytometry to distinguish between apoptotic cells and normal cells,thereby obtaining the effect of drugs on cell apoptosis.According to the experimental results,SQFZ alone had no significant effect on the apoptosis of A549 cells.Cisplatin alone promoted the apoptosis of A549 cells,and the apoptosis promotion effect increased with the increased concentration of cisplatin,showing a concentration correlation effect.The combination use of SQFZ and different concentraions ofcisplatin showed significant synergistic effect on promoting cell apoptosis.According to the results of network pharmacology analysis and apoptosis experiments,the potential synergistic anti-lung cancer mechanism was further verified by Western Blot Results showed that the combination of SQFZ and DDP could up-regulate theexpression of apoptosis-related protein Caspase-3,Caspase-9 and Bax/Bcl-2,and inhibite the expression of apoptosis-related proteins JAK2,STAT3,P65 and P50.Thus,it coulde be speculated that the mechanism of SQFZ combined with DDP to enhance the anti-lung cancer of cisplatin may be related to the enhanced expression of caspase-3,caspase-9,bax/bcl-2 protein and the inhibition of JAK-STAT and NF-kB pathways.3 Pharmacodynamic study of SQFZ combined with DDP on C57BL/6 mice subcutaneous lung cancer ModelThrough in vitro cell experiments,we found that SQFZ injection combined with cisplatin could enhance the anti-lung cancer effect.This section used the tumor-bearing animal model to further explore the anti-lung cancer effect of the combination of the two.Firstly,a mice model of subcutaneous lung cancer was established.The mice were randomly divided into four groups:blank group,model group,cisplatin alone group and Shenqi Fuzheng and Cisplatin combination group.After the mouse subcutaneous tumor was modeled for 7 days,the blank group and the model group were given saline(10 ml/kg·d);the cisplatin group was administered cisplatin(2 mg/kg·d)for 5 days;the combination group was administered with cisplatin 2 mg/kg·d(stopping administration after 5 days of continuous administration)and Shenfu Fuzheng 10ml/kg(Shenqi Fuzheng injection was continuously administered for 17 days).After 17 days,the mice were sacrificed and the tumours were removed.The evaluation indicators included tumor weight,tumor inhibition rate,spleen/thymus index,immune factor IL-2 and the apoptotic protein expression content of tumor immunohistochemical tissue section.According to the weight of tumor and the tumor inhibition rate,the tumor weight of cisplatin group was lower than that of the model group.Cisplatin combined with Shenqi Fuzheng could inhibit the further growth of tumor,indicating that Shenqi Fuzheng could enhance the efficacy of cisplatin.The spleen/thymus index and the immunoglobulin IL-2 content showed that the combination use of cisplatin and SQFZ could enhance the immunity of tumor-bearing mice.The results of immunohistochemistry showed that the expression of apoptosis protein Caspase-3 and Bcl-2/Bax were increased and the expression of VEGF was decreased in the tumor tissue sections of the combination group,.It indicated that the combination of SQFZ and DDP has synergistic anti-lung cancer effect.On the one hand,SQFZ combined with DDP played an anti-tumour effect by improving the immunity of tumor-bearing mice;on the other hand,SQFZ combined with DDP also promoted the expression of apoptotic proteins and inhibited tumor growth.In a word,the combination of SQFZ and DDP played the synergistic anti-lung cancer effect through"Fuzheng" and "Quxie".