Modified Biejia Jianwan decoction restrains PD-L1-mediated immune evasion through the HIF-1α/STAT3/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Modified Biejia Jianwan (M-BJJW), a Traditional Chinese Medicine (TCM) decoction, has exhibited great potential in treating hepatocellular carcinoma (HCC). However, its underlying functional mechanism still remains unknown. AIM OF THE STUDY: The study aimed to explore the anti-hepatocarcinogenic effects of M-BJJW, specifically its influence on PD-L1-mediated immune evasion in hypoxic conditions, and elucidate the related molecular mechanisms in HCC. MATERIALS AND METHODS: To investigate the therapeutic efficacy and mechanisms underlying M-BJJW's effects on HCC, we employed a diethylnitrosamine (DEN)-induced rat model maintained for 120 days. Following model establishment, flow cytometry was utilized to assess the distribution of immune cell populations in peripheral blood, spleens, and tumor tissues after M-BJJW administration. Simultaneously, enzyme-linked immunosorbent assays (ELISA) were conducted to analyze cytokine profiles in serum samples. Immunohistochemistry was employed to determine the expression levels of crucial proteins within tumor tissues. Furthermore, HCC cells exposed to CoCl2 underwent Western blot analysis to validate the expression levels of HIF-1α, PD-L1, STAT3, and nuclear factor kappa B (NF-κB) p65. The modulatory effects of STAT3 and NF-κB p65 were investigated using specific inhibitors and activators in wild-type cell lines. High-performance liquid chromatography coupled with mass spectrometry (HPLC/MS) was utilized to identify the chemical constituents present in M-BJJW-medicated serum. The immunomodulatory properties and the anti-tumor activities of M-BJJW were evaluated by co-culturing with peripheral blood mononuclear cells (PBMC) and the CCK-8 assay. Additionally, we assessed M-BJJW's impact on hypoxia-induced alterations in HCC cell lines using immunofluorescence and Western blot assessments. RESULTS: M-BJJW exhibited substantial therapeutic advantages by effectively alleviating pathological deterioration within the HCC microenvironment. In the DEN-induced rat model, M-BJJW administration notably reduced tumor growth. Flow cytometry analyses revealed an increased proportion of Cytotoxic T lymphocytes (CTLs) accompanied by a simultaneous decrease in regulatory T cells (Tregs). ELISA data supported a marked decrease in pro-inflammatory cytokines, including interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor α (TNF-α). Immunohistochemistry confirmed the suppressive effect of M-BJJW on the expression of HIF-1α and PD-L1. Notably, western blotting unveiled the role of HIF-1α in regulating PD-L1 expression via the STAT3 and NF-κB signaling pathways in HCC cell lines, which was validated using activators and inhibitors of STAT3 and NF-κB. The CCK-8 assay and co-culture techniques demonstrated the anti-tumor activity of M-BJJW. Immunofluorescence and western blotting further confirmed that M-BJJW-containing serum dose-dependently inhibited HIF-1α, PD-L1, p-STAT3, and p-p65 in hypoxic HCC cell lines. CONCLUSIONS: M-BJJW demonstrates significant therapeutic potential against HCC by influencing the hypoxic microenvironment, thereby regulating the immunosuppressive milieu. Specifically, M-BJJW modulates the HIF-1α/STAT3/NF-κB signaling pathway, leading to reduced PD-L1 expression and an elevated ratio of cytotoxic T lymphocytes (CTLs), while concurrently decreasing T regulatory cells (Tregs) and immunosuppressive factors. These synergistic effects aid in countering PD-L1-mediated immune evasion, presenting compelling pharmacological evidence supporting the clinical application of M-BJJW as a therapeutic approach for HCC.

Mechanism Exploration of Euphorbia fischeriana Steud. for Liver Cancer Based on Aspartic Acid Identification in Metabolomics.

OBJECTIVE: To investigate the anti-liver cancer effects and aspartic acid (Asp)-related action mechanism of Euphorbia fischeriana Steud. (Lang Du, LD). METHODS: The mice model of liver cancer was established by injection of H22 cells. After 5 days, mice were randomly divided into model group, sorafenib group (20 mg/kg), LD high-dose (LDH, 1.36 g/kg) group, LD medium-dose (LDM, 0.68 g/kg) group, and LD low-dose (LDL, 0.34 g/kg) group, 10 mice each group. Drugs were intragastrically administered to the mice once daily for 10 days, respectively. Body weight, tumor size and tumor weight were recorded. Hepatic index was calculated. Pathological changes of liver cancer tissues were evaluated by hematoxylin and eosin staining and TUNEL staining. Liquid chromatography-mass spectrometer was used to analyze different metabolites between the model and LDH groups. RESULTS: After LD treatment, tumor weight, tumor size and hepatic index were reduced compared with the model group. Necrocytosis and karyorrhexis of tumor cells were found. Moreover, 61 differential metabolites (18 up-regulated, 43 down-regulated) were affirmed and 20 pathways of KEGG (P<0.05) were gotten. In addition, Bel-7402, HepG2 and H22 cell viabilities were significantly increased after adding Asp into the medium. And then, the cell proliferation effect induced by Asp was ameliorated by LD. CONCLUSION: The anti-liver cancer efficacy of LD extract was validated in H22 mice model, and inhibition of Asp level might be the underlying mechanism.

Tribulus terrestris L. induces cell apoptosis of breast cancer by regulating sphingolipid metabolism signaling pathways.

BACKGROUND: Tribulus terrestris L. (TT) was initially documented in Shen-Nong-Ben-Cao-Jing and has been used for thousands of years in China as a herb to calm liver, dispel melancholy and wind, promote blood circulation, improve eyesight, and relieve itching. Moreover, it was also used to treat breast cancer in ancient China. However, the pharmacological activities of TT extract on breast cancer have received little attention. PURPOSE: In this study, we investigated the anti-breast cancer effects and possible mechanisms of action of this herbal drug. METHODS: Network pharmacology analysis the study of network pharmacology was done to analyze the possibility of TT's anti-breast cancer effect. And then, molecular docking between TT7/TT8 and vascular endothelial growth factor receptor 2 (VEGFR2) were performed by Autodock software as well as the related protein expressions were analyzed by western blot to verify this effect. In vivo experiment: The mouse model of breast cancer was established by injection of 4T1 cells. Then drugs were intragastrically administered to the mice once daily for fourteen days. Body weight, tumor size, and tumor weight were recorded at the end of the experiment. Moreover, tumor inhibitory rate was calculated. Finally, pathological changes and apoptosis of breast cancer tissues were respectively evaluated by HE and Hoechst staining. Proteomics and metabonomics analyses: The tumor tissues were chosen to perform conjoint analysis. Firstly, differential proteins and metabolites were found. Furthermore, the functional analyses of them were analyzed by software. At the last, immunofluorescent staining of SGPP1, SPHK1 and p-SPHK1 in tumor tissue were done. RESULTS: 12 active ingredients of TT, 127 targets of active ingredients, 15,253 targets of breast cancer, 1,225 targets of Ru yan, and 123 overlapping genes were obtained in the network pharmacology study. There was firm conjunction between TT7/TT8 and VEGFR2. Besides, tumor size and weight were markedly reduced in TT groups compared to the model group. The tumor inhibitory rate was more than 26% in TTM group. After drug treatment, many adipocytes and cracks between tumor and apoptosis were discovered. The western blot results showed that TT aqueous extract lowered the levels of VEGFR2, ERK1/2, p-ERK1/2 (Thr202, Tyr204) and Bcl2, while increasing the levels of Bax and the ratio of Bax/Bcl2. Furthermore, 495 differential proteins and 76 differential metabolites were found between TTM and model groups with the sphingolipid metabolism pathway being enriched. At last, TT treatment significantly reduced the levels of SGPP1, SPHK1 and p-SPHK1 in tumor tissue. CONCLUSIONS: In conclusion, TT demonstrates therapeutic effects in a mouse model of breast cancer, and its mechanism of action involves the regulations of sphingolipid metabolism signaling pathways. This study lends credence to the pharmacological potential of TT extract as a breast cancer therapy.

Promoting liver cancer cell apoptosis effect of Tribulus terrestris L. via reducing sphingosine level was confirmed by network pharmacology with metabolomics.

Background: Tribulus terrestris L. (TT) is one of the most common Chinese herbs and distributes in various regions in China. TT was first documented to treat breast cancer in Shen-Nong-Ben-Cao-Jing. However, the pharmacological activities of TT extract on liver cancer have not been reported. In this study, we investigated its anti-liver cancer activity and underlying mechanism. Methods: Traditional Chinese Medicine Systems Pharmacology (TCMSP) and PharmMapper databases were used to obtain the active ingredients and the targets of TT. Genecards database was employed to acquire TT targets in liver cancer. Furthermore, Venny 2.1, Cytoscape 3.8.2, DAVID 6.8 software were utilized to analyze the relationship between TT and liver cancer. In vivo experiment: The animal model of liver cancer was established by injection of H22 cells into Balb/c mice. After five days, drugs were intragastrically administered to the mice daily for 10 days. Body weight, tumor size and tumor weight were recorded. Tumor inhibitory rate was calculated. Protein levels were examined by Western blotting. Pathological changes of liver cancer tissues were evaluated by HE and Tunel staining. Metabolomics study: LC-MS was used to analyze different metabolites between model and TTM groups. Results: 12 active ingredients of TT, 127 targets of active ingredients, 17,378 targets of liver cancer, and 125 overlapping genes were obtained. And then, 118 items of GO biological processes (BP), 54 items of GO molecular function (MF), 35 items of GO cellular component (CC) and 128 pathways of KEGG were gotten (P < 0.05). Moreover, 47 differential metabolites were affirmed and 66 pathways of KEGG (P < 0.05) were obtained. In addition, after TT and sorafenib treatment, tumor size was markedly reduced, respectively, compared with model group. Tumor weight was significantly decreased and tumor inhibitory rate was more than 44% in TTM group. After TT treatment, many adipocytes, cracks between tumor cells and apoptosis were found. The levels of pro-Cathepsin B, Cathepsin B, Bax, Bax/Bcl2, Caspase3 and Caspase7 were markedly increased, but the level of Bcl2 was significantly reduced after TT treatment. Conclusion: TT has a broad range of effects on various signaling pathways and biological processes, including the regulation of apoptosis. It exhibits antitumor activity in an animal model of liver cancer and activates the apoptotic pathway by decreasing Sph level. This study provides valuable information regarding the potential use of TT extract in the treatment of liver cancer and highlights the importance of investigating the underlying molecular mechanisms of traditional medicines for the development of new therapeutic drugs in liver cancer.

Molecular and metabolic mechanisms of bufalin against lung adenocarcinoma: New and comprehensive evidences from network pharmacology, metabolomics and molecular biology experiment.

BACKGROUND: This study aims to evaluate the efficacy and therapeutic mechanism of bufalin on lung adenocarcinoma (LUAD) through a comprehensive strategy integrating network pharmacology, metabolomics and molecular biology verification. METHODS: The putative targets of bufalin were discerned from PharmMapper and Swiss Target Prediction database. LUAD-related targets were obtained by target filtering of GeneCard database and data mining of GEO database. PPI network was constructed to screen the core targets, and their clinical significance was assessed through several public databases. GO and KEGG pathway analyses were performed to identify possible enrichment of genes with specific biological themes. Molecular docking and molecular dynamics (MD) simulation were employed to determine the correlation and binding pattern between bufalin and core targets. The potential mechanisms of bufalin acting on LUAD, as predicted by network pharmacology analyses, were experimentally validated using in-vitro and in-vivo models. Finally, the effects of bufalin intervention on metabolite profile and metabolic pathway in LUAD nude mice were investigated by non-targeted metabolomics. RESULTS: 209 bufalin targets and 1082 LUAD-associated targets were harvested, of which 51 intersection targets were identified. 10 core targets including Akt1, STAT3, EGFR, CASP3 and SRC were picked out through network topology analysis, and they had a potent binding activity with bufalin as indicated by molecular docking and MD simulation. Hub module of PPI network was closely related to cell proliferation and apoptosis. GO and KEGG enrichment analyses suggested that bufalin exerted therapeutic effects on LUAD possibly by inhibiting proliferation and promoting apoptosis via PI3K/Akt, FoxO1 and MAPK/ERK pathways, which were confirmed by a series of in-vitro studies as well as HE, TUNEL and Ki-67 staining of tumor tissues. Further metabolomics analysis revealed that bufalin mainly regulated ABC transporter and remodeled AA metabolism, thereby contributing to the treatment of LUAD. CONCLUSION: From molecular and metabolic perspective, the present study not only provided a unique insight into the possible mechanisms of bufalin against LUAD after successfully filtering out associated key target genes, differential endogenous metabolites, and signaling pathways, but also proposed a novel promising therapeutic strategy for LUAD.

Preparation and characterization of parthenolide nanocrystals for enhancing therapeutic effects of sorafenib against advanced hepatocellular carcinoma.

A novel nanocrystals delivery system of parthenolide (PTL) was designed to combined application with sorafenib (Sora) for advanced hepatocellular carcinoma (HCC) therapy, attempting to not only improve the poor aqueous solubility of PTL, but also enhance the synergistic therapeutic effects with Sora. The PTL nanocrystals (PTL-NCs) were prepared by precipitation-high-pressure homogenization method. The formed PTL-NCs with rod morphology possessed size of 126.9 ± 2.31 nm, zeta potential of -11.18 ± 0.59 mV and drug loading of 31.11 ± 1.99%. Meanwhile, PTL in PTL-NCs exhibited excellent storage stability and sustained release behavior. The combination therapy of Sora and PTL-NCs (Sora/PTL-NCs) in vitro for HepG2 cells presented superior therapeutic effects over that of individual PTL and Sora on intracellular uptake, cell proliferation inhibition and migration inhibition. Meanwhile the strongest anti-tumor effect with 81.86% inhibition rate and minimized systemic toxicity of Sora/PTL-NCs in vivo were obtained on tumor-bearing mice compared with that of PTL (48.84%) and Sora (58.83%). Thus, these findings suggested that PTL-NCs as an effective delivery system for the synergistically used with Sora to gain an optimal response against HCC, for referenced in the industrialization of nanocrystals products for intravenous administration.

Adjuvant effects of fermented red ginseng extract on advanced non-small cell lung cancer patients treated with chemotherapy.

OBJECTIVE: To investigate the adjuvant therapeutic effects of fermented red ginseng (FRG) extract on non-small cell lung cancer (NSCLC) patients treated with chemotherapy. METHODS: A total of 60 patients with advanced NSCLC were divided into two groups using a random number table, i.e., the gemcitabine plus cisplatin (GP) chemotherapy alone group (26 patients) and the FRG + GP chemotherapy group (34 patients), for 60-day treatment. Patients were then assessed according to the Fatigue Symptom Inventory, Chinese medicine symptoms score, Self-Rating Anxiety Scale, Self-Rating Depression Scale, Karnofsky Performance Status Scale, and Functional Assessment of Cancer Therapy-Lung. In addition, chemotherapy toxicity and tumor biomarkers were measured. RESULTS: For NSCLC patients after chemotherapy, FRG extract significantly improved the FSI score, CM symptoms score, psychological status, physical conditions, and quality of life and reduced chemotherapy toxicity, but the expression levels of carcinoembryonic antigen, cytokeratin-19 fragments, and neuron-specific enolase were not significantly different between the chemotherapy alone and the FRG + chemotherapy groups or between pre- and post-treatments. CONCLUSIONS: This study demonstrated that FRG extract had an adjuvant effect on advanced NSCLC patients treated with chemotherapy. Further studies with a larger sample size will verify the current findings.

[Prevention and treatment of pulmonary-fibrosis by traditional Chinese medicine].

Pulmonary-fibrosis (PF) is the result of interstitial lung disease which has different causes and it is one of the most intractable diseases in respiratory system. The PF is characterized by progressive difficulty of breath and dying of respiratory failure, seriously threatening the health of people. Immunosuppressive drugs and glucocorticoids have been widely adopted as the main methods in treatment, but the results are not satisfactory and the side effects are obvious. Many specialists have tried traditional Chinese medicine in recent years with satisfactory effect, indicating that the prospect of using traditional Chinese medicine in the treatment of PF is optimistic. This article outlines the researches about using traditional Chinese medicine in the treatment of PF.