Hsa_circRNA_007630 knockdown delays colon cancer progression by modulation of ferroptosis via miR-506-3p/AURKA axis.

Colon cancer contributes to high mortality rates internationally that has seriously endangered human health. Aurora kinase A (AURKA) served as a key molecule in colon cancer. However, its role of AURKA on regulating ferroptosis in colon cancer and their possible interactions with miRNAs and circRNAs remain still elusive. Comprehensive bioinformatics analysis after RNA-sequencing was conducted to determine the differentially expressed genes (DEGs), ferroptosis-related DEGs and hub genes. The direct relationship between miR-506-3p and hsa_circRNA_007630 or AURKA was predicted, then verified by dual luciferase reporter and quantitative real-time polymerase chain reaction. The rescue experiments were conducted by cotransfection with si-hsa_circRNA_007630, miR-506-3p inhibitor or pcDNA-AURKA in HT29 cells. Erastin was used to induce ferroptosis in HT29 cells and validated by detecting levels of intracellular Fe2+, lipid reactive oxygen species, glutathione, malondialdehyde and ferroptosis markers expression. We screened a total of 331 DEGs, 26 ferroptosis-related genes, among which 3 hub genes were identified through PPI network analysis. Therein, AURKA expression was elevated in colon cancer cells. Moreover, AURKA was targeted by miR-506-3p, and hsa_circRNA_007630 operated as miR-506-3p sponge. The effect of hsa_circRNA_007630 depletion on the inhibiting malignant phenotypes of HT29 cells was rescued by inhibition of miR-506-3p or AURKA overexpression. Additionally, AURKA reduced erastin-induced ferroptosis in HT29 cells. Depletion of circRNA_007630 exerts as a suppressive role in colon cancer through a novel miR-506-3p/AURKA pathway related to ferroptosis, and might become a novel marker for colon cancer.

Tumor Microenvironment following Gemcitabine Treatment Favors Differentiation of Immunosuppressive Ly6Chigh Myeloid Cells.

Regulation of myeloid-derived suppressor cells (MDSC) by ongoing inflammation following repeated chemotherapy remain elusive. In this study, we show that a multidose clinical regimen of gemcitabine (GEM) treatment enhances the immunosuppressive function of monocytic MDSC (M-MDSC), although tumor development is delayed in E0771 tumor-bearing mice. Accordingly, effector IFN-γ-producing CD4 and CD8 T cells are significantly decreased in the tumor microenvironment (TME) of GEM-treated mice. The conditioned medium of GEM-treated tumor cells enhances differentiation of mouse bone marrow cells and human PBMC into immunosuppressive M-MDSC. Cytokine profiling of GEM-treated tumor cells identifies GM-CSF as one of the most differentially expressed cytokines. Blockade or knockdown of GM-CSF can partially reduce immunosuppression of Ly6Chigh cells induced by GEM-conditioned medium. Knockdown of GM-CSF in tumor cells also delays tumor progression with decreased accumulation of M-MDSC in the TME. Mechanistically, enhanced production of reactive oxygen species and activation of NF-κB are observed in GEM-treated tumor cells. Treatment with the mitochondrial-targeted antioxidant and inhibitor of NF-κB signaling can abrogate GEM-induced hyperexpression of GM-CSF in E0771 cells. In addition, the phagocytic clearance of apoptotic tumor cells (efferocytosis) enhances the immunosuppressive function of bone marrow Ly6Chigh myeloid cells. Further, GEM treatment results in metabolic changes in residual tumor cells, leading to the resistance to T cell-mediated killing. Together, our results define an undesired effect of repeated GEM treatment promoting immunosuppression in TME via upregulation of GM-CSF and efferocytosis as well as deregulation of lipid metabolism in residual tumor cells.

[Composition and immunomodulatory activity of neutral and acidic polysaccharides isolated from Epimedii Folium].

Epimedii Folium possesses many pharmacological activities including immunomodulation, anti-oxidation, and anti-tumor. Polysaccharides are the main components of Epimedii Folium, and their activities are closely related to the structure. The present study isolated a neutral polysaccharide(EPS-1-1) and an acidic polysaccharide(EPS-2-1) from the aqueous extract of Epimedii Folium through DEAE-52 cellulose anion-exchange chromatography and Sephadex G-100. The structures were characterized by chemical composition analysis, high-performance gel permeation chromatography(HPGPC), Fourier-transform infrared spectrometry(FT-IR), 1-phenyl-3-methyl-5-pyrazolone(PMP) derivatization, scanning electron microscopy(SEM), Congo red test, etc. The immunomodulatory activity of polysaccharides in vitro was determined by investigating the effects on the maturation of bone marrow-derived dendritic cells(BMDCs) and the release of inflammatory cytokines. According to the structural characterization analysis, EPS-1-1 was composed of fructose(Fuc), mannose(Man), ribose(Rib), rhamnose(Rha), glucose(Glc), galactose(Gal), xylose(Xyl), and arabinose(Ara) at 1.90∶0.67∶0.05∶0.08∶3.29∶1.51∶0.05∶0.37(molar ratio), while EPS-2-1 was mainly composed of Fuc, Man, Rha, glucuronic acid(GlcA), galacturonic acid(GalA), Glc, Gal, Xyl, and Ara at 5.25∶0.18∶0.32∶0.13∶1.14∶0.16∶0.55∶0.08∶0.2. EPS-1-1 and EPS-2-1 could promote the maturation and function of BMDCs through up-regulating the expression of MHC-Ⅱ, CD86, CD80, and CD40, and increasing the levels of inflammatory cytokines(IL-6, IL-12, and TNF-α) in vitro experiments, which suggested that EPS-1-1 and EPS-2-1 possessed good immunomodulatory activity.

[Effects and mechanism of epimedium polysaccharide on solubility of icariin and baohuoside â ].

This study evaluated the effects of epimedium polysaccharide(EPS) on the solubility of icariin and baohuoside Ⅰ so as to preliminary explore its solubilization function and the underlying mechanism. The solubility of these two insoluble flavonoids in water and polysaccharide solutions was compared by high performance liquid chromatography, and the mechanism was investigated by diffe-rential scanning calorimetry(DSC) and critical micelle concentration determination. The results indicated that their solubilization in crude EPS solutions was concentration-dependent. The solubility of icariin and baohuoside Ⅰ in 20 mg·mL~(-1) EPS-1-1 was 9.05 times and 5.76 times that in water, respectively; while their solubility in 20 mg·mL~(-1) EPS-2-1 was 10.55 and 8.39 times that in water, respectively. The change of the DSC thermograms suggested the formation of new complexes from icariin and baohuoside Ⅰ with polysaccharides. The critical micelle concentrations proved the micellar properties of both EPS-1-1 and EPS-2-1. In short, EPS can significantly increase the solubility of icariin and baohuoside Ⅰ, the mechanism of which may be related to the formation of micellar complexes between EPS and insoluble flavonoids.

Circular RNA-0001283 Suppresses Breast Cancer Proliferation and Invasion via MiR-187/HIPK3 Axis.

BACKGROUND Circular RNAs (circRNAs) are key regulators that take part in the carcinogenesis and development of breast cancer. The current study aimed to identify the expression of and explored the function of circRNA-0001283 in breast cancer. MATERIAL AND METHODS Breast cancer tissue samples were tested using high-throughput sequencing to identify the levels of relative genes; and proteins were addressed by using quantitative real-time polymerase chain reaction (qRT-PCR) and western-blot. Cell ability and cell apoptosis were investigated by Cell Counting Kit-8 (CCK-8) and flow cytometry. Invasion was detected by Transwell invasion assay. The identification of target genes was analyzed by dual-luciferase reporter assay. RESULTS Downregulation of circRNA-0001283 expression was observed in breast cancer tissue samples. Ectopic expression of circRNA-0001283 remarkably suppressed cell viability and invasion, and induced apoptosis in breast cancer cells. Furthermore, circRNA-0001283 bound to miR-187 and decreased the expression of miR-187, which resulted in inhibition in cell growth and invasion. Finally, we showed that circRNA-0001283 positively regulated HIPK3 expression by sponging miR-187. CONCLUSIONS The results reveal a new functional circRNA-0001283 in breast cancer and may provide targets for developing novel therapeutic strategies for breast cancer.

Regulation of Eclipta prostrata L. components on cigarette smoking-induced autophagy of bronchial epithelial cells via keap1-Nrf2 pathway.

Cigarette smoking extract (CSE)-induced autophagic injury has been regarded as an important contributor to the pathogenesis of lung cancer. We previously found that Eclipta prostrata L. component (CCE) reduced CSE-induced bronchial epithelial cells damage. However, the mechanism remains unknown. Human normal bronchial epithelial cells (NHBE) were exposed to CSE to establish stress model. Nrf2-siRNA and Keap1-siRNA transfection were performed. mRFP-GFP-LC3 dual fluorescence and transmission electron microscopy were used to observe the autophagic characteristics. CCE prevented CSE-induced Nrf2 transfer into cytoplasm and up-regulated Keap1 level of NHBE cells. Furthermore, CCE significantly increased p-p16, p-p21 and p-p53 phosphorylation levels in Nrf2-siRNA- or Keap1-siRNA-transfected cells. As demonstrated by transmission electron microscopy and mRFP-GFP-LC3 dual fluorescence assays, CCE mitigated autophagic injury, and also down-regulated autophagy-related Beclin-1, LC3II/LC3I ratio, Atg5 and ATF4 levels. Our findings showed the attenuation of CCE on CSE-induced NHBE cells injury was associated with Nrf-2-mediated oxidative signaling pathway.

[Response surface analysis for the optimization of extraction condition for polysaccharides from Epimedium polysaccharides and studies on its tumor immune activities].

To enhance the efficiency of the extraction for Epimedium koreanum Nakai polysaccharides, we conducted the Box-Benhnken experiment and examined the response surface. C57 BL6 mice were used as Lewis-bearing mice model in the study of the polysaccharides in the regulation of tumor immunity. In the first place, different factors including extraction time, temperature, and ratio were evaluated in the yield of polysaccharides. The second order polynomial equation was formed to fit the experimental data. The optimal condition was temperature 87 ℃, extraction time 3.84 h, and ratio of solution and material 1:16.33 to get polysaccharides from Epimedium koreanum Nakai. Under this condition, model-predicted and experimentally measured values of polysaccharide yield were 1.045% and 1.023%, respectively, with a relative error between them at 2.15%. The extraction method is reliable, simple and applicable to the extraction of Epimedium polysaccharides. In addition, this data suggests that Epimedium polysaccharides have obvious immune-regulatory activities in the tumor-bearing mice through increasing the immune-enhancing cytokines to override the immune- suppressing factors.

[Research thoughts on tumor immune responses by polysaccharide of Chinese medicine via oral administration].

Tumor immunotherapy is one of the most significant scientific progresses. The idea of applying the traditional Chinese theory of "the balance of Yin and Yang" to treat cancer is in accordance with that of modern tumor immune strategy. Researches indicated that polysaccharide of Chinese medicine through regulation in immune responses could offer better paradigm for tumor immune treatment under the traditional Chinese theory. However, current studies related to tumor immunotherapy largely focus on the immunity enhancement while lack of the exploration of suppressive factors. Meanwhile, the complex analysis and detection on composition as well as structure definitely increase the difficulty in mechanism of oral absorption and function in vivo. To better exploit novel Chinese medicine of polysaccharide for tumor immune treatment, this article will provide some constructive thoughts on regulation of tumor immune responses based on up to date researches of structure-function relationship, absorbent process and molecular mechanisms responsible for tumor immune as well.

[Research thoughts on structural components of Chinese medicine combined with bioinformatics].

Traditional Chinese medicine(TCM) is a complex system, featured with integrity and characteristics. Structural component TCM is a well-organized integrity of traditional Chinese medicine, reflecting multi-component integration effect of TCM. It gives us a new view on the material basis of TCM. Currently, conventional researching strategies are not enough to deal with the relationship between material basis and efficacy, multi-composition, multi-targets, and multi-section mechanism. Post-genome area gives a birth to bioinformatics, which involves systematic biology, different levels of omics, corresponding mathematics and computer techniques. It increasingly becomes a powerful tool to understand complicated system and life essential laws. Research ideas, methods. and knowledge of data mining technology of bioinformatics combined with the theory of structural components of Chinese medicine bring a new opportunity for developing structural components of Chinese medicine, systematically exploring the essence of TCM and promoting the modernization of TCM.

Metabolite profiles of epimedin C in rat plasma and bile by ultra-performance liquid chromatography coupled with quadrupole-TOF-MS.

Epimedin C is one of the major bioactive constituents of Herba Epimedii. In this study, the metabolite profiles of epimedin C in rat plasma and bile were qualitatively investigated, and the possible metabolic pathways of epimedin C were subsequently proposed. After oral administration of epimedin C at a single dose of 80 mg/kg, rat biological samples were collected and pretreated by protein precipitation. Then these pretreated samples were injected into an Acquity UPLC BEH C18 column and detected by ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry. In all, 12 metabolites were identified in the biosamples. Of these, eight, two from plasma and six from bile, are, to our knowledge, reported here for the first time. The results indicated that epimedin C was metabolized via desugarization, dehydrogenation, hydrogenation, dehydroxylation, hydroxylation, demethylation and glucuronidation pathways in vivo. Thus, this study revealed the possible metabolite profiles of epimedin C in rat plasma and bile.

[Comparison of protective effects of eight ethyl acetate extracts from Eclipta prostrate on NHBE cells based on component structure theory].

To analyze and compare the protective effects of active components in different ethyl acetate extracts (EAEEPs) from Eclipta prostrate, in order to study the comparison of materials bases protecting normal human bronchial epithelial (NHBE) cells. The MTT assay was taken to compare the protective effect of different EAEEPs on cigarette smoke extracts (CSE) -induced NHBE cells. The ultra-performance liquid chromatography (UPLC) was applied to analyze the content of phenolic acid, coumaric grass ether and flavonoid in EAEEPs. According to the results, all of the eight EAEEPs (0-200 mg x L(-1)) showed certain protective effect on NHBE cells, with statistical difference. Specifically, the total mass of EAEEP VII (89.15 mg x L(-1)) and EAEEP VIII (57.44 mg x L(-1)), which showed the strongest activity, was not the highest, while EAEEP III (132.25 mg x L(-1)) displayed the highest total mass. In the combination with the "component structure" theory, the analysis showed a significant difference in the mass structure among phenolic acid, coumaric grass ether and flavonoid in EAEEP VIII and EAEEP VIII, which were 1.0: 1. 0: 0.5 and 1.0: 1.9: 0.8, respectively. The results suggested a specific optimal "component structure" relationship may exist in EAEEP, which could provide reference for the material base study and quality control.

[Study on oral absorption enhancers of astragalus polysaccharides].

Astragalus polysaccharides was lounded to 4-(2-aminoethylphenol), followed by labeling the APS-Tyr with fluorescein-5-isothiocyanate (FITC) at the secondary amino group. The absorption enhancement effects of low molecular weight chitosan and protamine on astragalus polysaccharides were evaluated via Caco-2 cell culture model. The results show that the fluorecent labeling compound has good stability and high sensitivity. On the other hand low molecular weight chitosan and protamine also can promoted absorption of the astragalus polysaccharides without any cytotoxity, and the absorption increase was more significant with increasing the amount of low molecular weight chitosan and protamine. At the same time, the low molecular weight chitosan has slightly better effect. The transepithelial electric resistance (TEER) of Caco-2 cells show that absorption enhancers could improve its membrane transport permeability by opening tight junctions between cells and increasing the cell membrane fluidity.

[Construction of research system for processing mechanism of traditional Chinese medicine based on chemical composition transformation combined with intestinal absorption barrier].

Based on practice of Epimedium processing mechanism for many years and integrated multidisciplinary theory and technology, this paper initially constructs the research system for processing mechanism of traditional Chinese medicine based on chemical composition transformation combined with intestinal absorption barrier, which to form an innovative research mode of the " chemical composition changes-biological transformation-metabolism in vitro and in vivo-intestinal absorption-pharmacokinetic combined pharmacodynamic-pharmacodynamic mechanism". Combined with specific examples of Epimedium and other Chinese herbal medicine processing mechanism, this paper also discusses the academic thoughts, research methods and key technologies of this research system, which will be conducive to systematically reveal the modem scientific connotation of traditional Chinese medicine processing, and enrich the theory of Chinese herbal medicine processing.

Et2 Zn-Mediated Gem-Dicarboxylation of Cyclopropanols with CO2.

An unprecedented Et2 Zn-mediated gem-dicarboxylation of C─C/C─H single bond of cyclopropanols with CO2 is disclosed, which provides a straightforward and efficient methodology for the synthesis of a variety of structurally diverse and useful malonic acids in moderate to excellent yields. The protocol features mild reaction conditions, excellent functional group compatibility, broad substrate scope, and facile derivatization of the products. DFT calculations confirm that the transition-metal-free transformation proceeds through a novel ring-opening/α-functionalization/ring-closing/ring-opening/ß-functionalization (ROFCOF) process, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) plays dual important roles in the transformation.

Ganoderma lucidum spores-derived particulate ß-glucan treatment improves antitumor response by regulating myeloid-derived suppressor cells in triple-negative breast cancer.

Granular ß-1,3-glucan extracted from the wall of Ganoderma lucidum spores, named GPG, is a bioregulator. In this study, we investigated the structural, thermal, and other physical properties of GPG. We determined whether GPG ameliorated immunosuppression caused by Gemcitabine (GEM) chemotherapy. Triple-negative breast cancer mice with GPG combined with GEM treatment had reduced tumor burdens. In addition, GEM treatment alone altered the tumor microenvironment(TME), including a reduction in antitumor T cells and a rise in myeloid-derived suppressor cells (MDSC) and regulatory T cells (Tregs). However, combined GPG treatment reversed the tumor immunosuppressive microenvironment induced by GEM. GPG inhibited bone marrow (BM)-derived MDSC differentiation and reversed MDSC expansion induced by conditioned medium (CM) in GEM-treated E0771 cells through a Dectin-1 pathway. In addition, GPG downgraded PD-L1 and IDO1 expression on MDSC while boosting MHC-II, CD86, TNF-α, and IL-6 expression. In conclusion, this study demonstrated that GPG could alleviate the adverse effects induced by GEM chemotherapy by regulating TME.

A novel drug-phospholipid complex loaded micelle for baohuoside I enhanced oral absorption: in vivo and in vivo evaluations.

Baohuoside I is an effective anti-cancer drug currently used for a variety of cancers in vitro. Unfortunately, baohuoside I has a very poor solubility in both water and in organic solvents. Besides that, it is subject to significant efflux. This work therefore aimed at evaluating the ability of mixed micelles to solubilize baohuoside I, increase permeability and inhibit efflux of baohuoside I to promote oral absorption. A novel (TPGS-baohuoside I-phospholipid complex) mixed micelles was formed by phospholipid complex and TPGS to increase the solubility, enhance permeability, and inhibit efflux of baohuoside I. Micelle formation was confirmed by differential scanning calorimetry and transmission electron microscopy. The average diameters and efflux ratio of mixed micelles decreased as the ratio of TPGS increased with a respective increase in solubility of baohuoside I. Nevertheless, a slow release of baohuoside I from loaded micelles was noted. The results showed that at a 1:9 ratio for baohuoside I-phospholipid complex: TPGS in mixed micelles, solubility of baohuoside I increased up to 88 fold while the efflux ratio decreased by 85%. Their smaller size and higher zeta potential values indicated that mixed micelles would be easily absorbed and more stable. The relative bioavailability of the micelles (AUC(0-∞)) compared with baohuoside I (AUC(0-∞)) was 533%, demonstrating great potential for clinical application. Hence, the novel micelles formed with phospholipid complex and TPGS considerably increased drug concentration in the media and enhanced absorption.

[Polybasic research on the biopharmaceutical characteristics of 20 (S)-protopanaxadiol].

In this study, the biopharmaceutical properties of 20 (S)-protopanaxadiol (PPD) were studied. Firstly, the equilibrium solubility and apparent oil/water partition coefficient of PPD were used to predict the absorption in vivo. Meanwhile the membrane permeability and absorption window were studied by Caco-2 cell model and single-pass intestinal perfusion model. Furthermore, the bioavailability and metabolism were combined to study the absorption properties and metabolic properties in vivo. All of them were used to provide theoretical and practical foundation for designing PPD preparation. The results showed that PPD is poorly water-soluble, and the equilibrium solubility in water is only 35.24 mg x L(-1). The oil-water partition coefficient is 46.21 (logP = 1.66). By Caco-2 cell model, the results showed PPD uptake in general, and it also has efflux. By in situ intestinal perfusion model, the results showed that the absorption of PPD in the intestine is good, and the effective permeability coefficient were duodenum > jejunum > ileum > colon. The oral bioavailability of PPD was 29.39%. It was not well. Metabolic studies showed PPD in vivo presented a wide spread metabolism. So the main factors that restricted oral bioavailability of PPD were the poor solubility and first-pass effect.

[Study on effective substances of tea for chemoprevention of lung cancer based on principal component analysis].

OBJECTIVE: To study the effective substances of tea for chemoprevention of lung cancer based on Principal Component Analysis (PCA). METHODS: UPLC was used to determine the content of each component in tea, MTT was used to analyze the survival of CSE-induced NHBE cells. DTNB and the colorimetric assay were used to detect the rate of GSH/GSSG, PCA was used to calculate the correlation coefficient matrix, eigen values and variance of contribution ratio to chemoprevention of lung cancer. RESULTS: Longjing (L) was the most effective one to protect NHBE cells from damaging, and the IC50 of L for prevention of NHBE cells was 0.2559 mg/mL and could make the GSH/GSSG ratio recovery ranging from 0.142 to 0.858 in a dose-dependent manner. The contribution of GCG prevention of lung cancer was 79.602% and that of GA was 11.037% by PCA. CONCLUSION: Different kinds of tea have different protective effect on NHBE cells. GCGC and GA are the main active ingredients in tea for chemoprevention of lung cancer by PCA.

Zengshengping improves lung cancer by regulating the intestinal barrier and intestinal microbiota.

Lung cancer is a common malignant tumor in clinical practice, and its morbidity and mortality are in the forefront of malignant tumors. Radiotherapy, chemotherapy, and surgical treatment play an important role in the treatment of lung cancer, however, radiotherapy has many complications and even causes partial loss of function, the recurrence rate after surgical resection is high, and the toxic and side effects of chemotherapy drugs are strong. Traditional Chinese medicine has played a huge role in the prognosis and improvement of lung cancer, among them, Zengshengping (ZSP) has the effect of preventing and treating lung cancer. Based on the "gut-lung axis" and from the perspective of "treating the lung from the intestine", the purpose of this study was to research the effect of Zengshengping on the intestinal physical, biological, and immune barriers, and explore its role in the prevention and treatment of lung cancer. The Lewis lung cancer and urethane-induced lung cancer models were established in C57BL/6 mice. The tumor, spleen, and thymus were weighed, and the inhibition rate, splenic and thymus indexes analyzed. Inflammatory factors and immunological indexes were detected by enzyme-linked immunosorbent assay. Collecting lung and colon tissues, hematoxylin and eosin staining was performed on lung, colon tissues to observe histopathological damage. Immunohistochemistry and Western blotting were carried out to detect tight junction protein expression in colon tissues and expression of Ki67 and p53 proteins in tumor tissues. Finally, the feces of mice were collected to investigate the changes in intestinal microbiota using 16SrDNA high-throughput sequencing technology. ZSP significantly reduced tumor weight and increased the splenic and thymus indexes. It decreased expression of Ki67 protein and increased expression of p53 protein. Compared with Model group, ZSP group reduced the serum levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor α (TNF-α), and ZSP group increased the concentration of secretory immunoglobulin A (sIgA) in the colon and the bronchoalveolar lavage fluid (BALF). ZSPH significantly increased the level of tight junction proteins such as ZO-1, Occludin and Claudin-1. Model group significantly reduced the relative abundance of Akkermansia (p < 0.05) and significantly promoted the amount of norank_f_Muribaculaceae, norank_f_Lachnospiraceae (p < 0.05) compared with that in the Normal group. However, ZSP groups increased in probiotic strains (Akkermansia) and decreased in pathogens (norank_f_Muribaculaceae, norank_f_Lachnospiraceae). Compared with the urethane-induced lung cancer mice, the results showed that ZSP significantly increased the diversity and richness of the intestinal microbiota in the Lewis lung cancer mice. ZSP played an important role in the prevention and treatment of lung cancer by enhancing immunity, protecting the intestinal mucosa and regulating the intestinal microbiota.

Integrating strategies of metabolomics, network pharmacology, and experiment validation to investigate the processing mechanism of Epimedium fried with suet oil to warm kidney and enhance yang.

Introduction: Epimedium, a traditional Chinese medicine (TCM) commonly used in ancient and modern China, is one of the traditional Chinese medicines clinically used to treat kidney yang deficiency syndrome (KYDS). There are differences in the efficacy of Epimedium before and after processing, and the effect of warming the kidney and enhancing yang is significantly enhanced after heating with suet oil. However, the active compounds, corresponding targets, metabolic pathways, and synergistic mechanism of frying Epimedium in suet oil to promote yang, remain unclear. Methods: Herein, a strategy based on comprehensive GC-TOF/MS metabolomics and network pharmacology analysis was used to construct an "active compounds-targets-metabolic pathways" network to identify the active compounds, targets and metabolic pathways involved. Subsequently, the targets in kidney tissue were further validated by real-time quantitative polymerase chain reaction (RT-qPCR). Histopathological analysis with physical and biochemical parameters were performed. Results: Fifteen biomarkers from urine and plasma, involving five known metabolic pathways related to kidney yang deficiency were screened. The network pharmacology results showed 37 active compounds (13 from Epimedium and 24 from suet oil), 159 targets, and 267 pathways with significant correlation. Importantly, integrated metabolomics and network pharmacologic analysis revealed 13 active compounds (nine from Epimedium and four from suet oil), 7 corresponding targets (ALDH2, ARG2, GSTA3, GSTM1, GSTM2, HPGDS, and NOS2), two metabolic pathways (glutathione metabolism, arginine and proline metabolism), and two biomarkers (Ornithine and 5-Oxoproline) associated with improved kidney yang deficiency by Epimedium fried with suet oil. Discussion: These finds may elucidate the underlying mechanism of yang enhancement via kidney warming effects. Our study indicated that the mechanism of action mainly involved oxidative stress and amino acid metabolism. Here, we demonstrated the novel strategies of integrating metabolomics and network pharmacology in exploring of the mechanisms of traditional Chinese medicines.