| Background:Lung cancer is a common clinical malignancy,with the second highest incidence rate and the highest mortality rate among malignancies worldwide.Non-small cell lung cancer(NSCLC)is the main type of lung cancer,and the development of new treatments and methods has been a major clinical challenge to be solved.Chinese medicine research on NSCLC has also been developing,and Xihuang Wan(XHW),as a classical anti-cancer Chinese medicine formula with definite clinical efficacy,has also played a role in the treatment of NSCLC.Previous studies have found that XHW can exert anti-tumor effects through various mechanisms such as induction of apoptosis,reversal of epithelial mesenchymal transformation(EMT),and regulation of immune microenvironment,but the studies were mostly focused on breast cancer and gastric cancer.For the mechanism of treatment of lung cancer,only the efficiency rate and tumor suppression rate of in vivo experiments are involved,which are relatively limited,therefore,there is still much room for the study of XHW in the treatment of NSCLC,which is worthy of further investigation.Objective:The present study combines systematic review,network pharmacology,in vitro experiments,in vivo experiments,and transcriptomics to study the mechanism related to the treatment of NSCLC by XHW,to provide more data support and scientific basis for XHW in the clinical treatment of NSCLC.Methods:1.We evaluated the effect of XHW in clinical randomized controlled trials for the treatment of NSCLC by systematic review and meta-analysis,and the evaluation indexes included:recent objective reponse,improvement of clinical symptoms,quality of life,and adverse effects.2.We predicted the active ingredients and main targets of XHW against NSCLC by network pharmacology.Firstly,all the active ingredients and corresponding predicted targets in XHW were retrieved through the Chinese medicine database BATMAN-TCM,and then NSCLC-related genes were retrieved through the disease databases DisGeNET,GeneCards,and OMIM.The two datasets were mapped,repeated targets were screened,to construct the"XHW-NSCLC-target" dataset.The dataset was then imported into String database for construcing protein-protein interactions network,and the obtained network was imported into Cytoscape 3.8.0 software for topological parameter analysis.The core targets of "XHW-NSCLC-target" were obtained.Then,the Metascape was used to annotate the biofunction of the core targets and analyze the main signaling pathways and biological processes of XHW against NSCLC.3.In vitro and in vivo studies were performed to verify XHW’s anti-NSCLC efficacy.In in vitro experiments,LL/2 cell line was used and the effects of XHW on the proliferation,apoptosis,cell cycle,migration ability and invasion ability were examined by CCK-8 assay,flow cytometry and Transwell assay.In in vivo experiments,C57/BL6 mice with Lewis lung cancer subcutaneous tumors were used,and the mice were divided into the control group,the low-dose XHW group(0.617 g/kg,by gavage,twice a day),the medium-dose XHW group(1.233 g/kg,by gavage,twice a day),the high-dose XHW group(2.466 g/kg,by gavage,twice a day),and the cisplatin group(3 mg/kg,intraperitoneal injection,once a week),and the blank group without tumor was also set.The general status,volume and weight of subcutaneous tumors,body weight and organ indexes of the mice were observed.The drugs were given for 15 days,and then the histopathological changes of tumors and liver and lung metastasis of subcutaneous tumors were observed by HE staining,and the effects on tumor proliferation were observed by Ki-67 immunohistochemical staining,and the spleen lymphocytes were detected by flow cytometry for CD3+,CD3+CD4+.CD3+CD8+and CD3+CD4+/CD3+CD8+immunophenotyping.4.Through transcriptomics methods,RNA sequencing was performed on the tumor tissues of the high-dose XHW group and the control group,differentially expressed genes were analyzed,and gne function annotation was performed to analyze the main signaling pathways and biological processes of XHW efficacy on Lewis lung carcinoma.5.Through polymerase chain reaction(PCR)and Western Blot(WB),the target molecules screened by network pharmacology and transcriptomics,including heat shock protein(HSP)family,estrogen receptor(ER),Retinoid X receptor(RXR),and peroxisome proliferator activated receptor(PPAR)were detected.Results:1.Meta analysis results showed that XHW as an adjuvant treatment of NSCLC could improve the short-term objective response,improve the patients’ clinical symptoms,improve the patients’ quality of life.However,in terms of improving liver and kidney dysfunction,improving gastrointestinal symptoms and blood system suppression,the adjuvant treatment of XHW did not have benefits.2.The network pharmacological analysis showed that XHW had 74 targets with predictable active ingredients,and the core action targets of "XHW-NSCLC-targets" totaled 230,including HSP90AA1,HSP90AB1,HSPAlA,ESR1,ESR2,RXRA,RXRB,RXRG,PPARG,PPARA,etc.The KEGG pathways associated with XHW’ s anti-NSCLC efficacy were pathways in cancer,PI3K-Akt signaling pathway,endocrine resistance,foxo signaling pathway,estrogen signaling pathway,EGFR tyrosine kinase inhibitor resistance,HIF-1 signaling pathway,T cell receptor signaling pathway,prolactin signaling pathway,Ras signaling pathway,focal adhesion,non-small cell lung cancer,etc.GO enrichment analysis showed that the main biological processes involved in XHW’s anti-NSCLC efficacy were cellular response to organic cyclic compounds,cellular response to hormonal stimulus,cellular response to lipids,apoptotic signaling pathway,response to steroid hormones,positive regulation of cell migration,positive regulation of cell death,positive regulation of cell motility,etc.3.The drug-containing serum of XHW did not have a significant proliferation inhibitory effect on Lewis lung cancer cell line LL/2 in vitro.XHW extract had a concentration-dependent inhibitory effect on LL/2,and the IC50 of XHW extract on LL/2 cells was 1.830 mg/mL at 24 h of action.The results of the flow cytometry showed that XHW extract had a significant pro-apoptotic effect on LL/2 cells at 24 h and could promote the transformation of LL/2 cell line to G2/M phase.Transwell assay showed that the effect of XHW extract significantly reduced the migration and invasion ability of LL/2 cell line at 24 h,and the effect was concentration-dependent.In a murine subcutaneous Lewis lung carcinoma model,the inhibitory effect of XHW on the growth of subcutaneous tumors was concentration-dependent,with the high-dose XHW group significantly inhibiting the growth of subcutaneous tumors in mice,and the dose was twice the equivalent dose in clinic.The pathological results of tumors showed that there were large areas of cell necrosis in the high-dose XHW group and the cisplatin group.Ki-67 immunohistochemical staining showed that both XHW and cisplatin significantly reduced the number of Ki-67-positive tumor cells,and this effect of XHW was not related to the concentrationThe pathological results of the liver showed normal liver parenchyma with no significant abnormalities in all groups.The results of splenocyte flow cytometry showed that no statistically significant differences were observed in CD3+、CD3+CD4+、CD3+CD8+and CD3+CD4+/CD3+CD8+spleen lymphocytes in all groups of mice.4.Transcriptomic sequencing of subcutaneous tumors in the high-dose XHW and control groups resulted in the screening of 406 differentially expressed genes,of which 130 were up-regulated and 276 were down-regulated in expression.KEGG pathway enrichment analysis of the differential genes showed that signaling pathways significantly altered were:cardiac muscle contraction,TNF signaling pathway,estrogen signaling pathway,adrenergic signaling in cardiomyocytes,insulin secretion,cyclic guanosine monophosphate-dependent protein kinase(cGMP-PKG)signaling pathway,regulation of lipolysis in adipocytes,apelin signaling pathway,proximal tubule bicarbonate reclamation,oxytocin signaling pathway,gastric acid secretion,salivary secretion,PPAR signaling pathway,calcium signaling pathway,and vascular smooth muscle contraction5.The results of PCR and WB showed that XHW significantly reduced the mRNA expressions of Hspb6,Hspb7,Hspbl,Hspb2,Hspala,Hspa8,Hsphl and the protein expressions of HSPB7,HSP27,HSP70 and HSPH1.The protein expression of HSC70 increased significantly,while the protein expression of HSP20 and HSP90 did not change significantlyXHW significantly reduced the mRNA expressions of Esr1,Rxra,Krt19,Ppara,Ppard and Pparg,decreased the protein expression of ER and RXR-α,and increased the protein expressions of PPARδ and PPARγ levels significantly,with no significant effect on the protein expression of KRT19 and PPARα.Conclusions:1.XHW as an adjuvant therapy for NSCLC could improve the recent objective response,improve patients’ clinical symptoms,improve patients’ quality of life.2.Network pharmacology experiments and transcriptomics experiments showed that XHW acts on NSCLC through multiple targets and pathways,and the core targets included HSP family and ER and so on.3.XHW showed efficacy in treating Lewis lung carcinoma in both in vitro and in vivo experiments,with no significant toxicity in the in vivo experiments,the effective dose was twice the equivalent dose in clinic.XHW’s anti-NSCLC mechanism might be related to reducing the mRNA and protein expression levels of HSP27,HSP70,HSPH1,ER and elevating the protein expression level of PPARy. |
