Jianpi Jiedu decoction suppresses colorectal cancer growth by inhibiting M2 polarization of TAMs through the tryptophan metabolism-AhR pathway.

BACKGROUND: Traditional Chinese medicine, JianpiJiedu decoction (JPJDF), has been utilized in colorectal cancer (CRC) treatment for over forty years. The potential of JPJDF to inhibit CRC through modulation of intestinal microbiota and their metabolites remains uncertain. AIMS: This study aims to further investigate the therapeutic mechanisms of JPJDF in CRC. METHODS: CAC mouse models were developed using azoxymethane (AOM) and dextran sulfate sodium (DSS). Intestinal tissues and contents underwent 16S rRNA gene sequencing and untargeted metabolomics analysis. Serum levels of IL-1ß and TNF-α were measured using ELISA. Immunohistochemistry was utilized to assess the expression of Ki67, ZO-1, Occludin, CD68, and CD206. Furthermore, western blotting was performed to evaluate the protein expression of AhR and NF-κB. RESULTS: JPJDF inhibited colorectal tumourigenesis in AOM/DSS treated mice, while also suppressing tumor cell proliferation and upregulating the expression of tight junction proteins. The results of 16S rRNA gene sequencing analysis revealed that JPJDF altered intestinal microbiota composition by increasing the abundance of beneficial bacteria. Additionally, JPJDF reduced tryptophan metabolites, effectively alleviating inflammation and significantly restoring intestinal barrier function in CAC mice. Molecular biology experiments confirmed that JPJDF suppressed the expression levels of AhR and M2-type tumor-associated macrophages, thereby promoting anti-tumor immunity and exerting inhibitory effects on CAC growth. CONCLUSION: JPJDF can regulate the tryptophan metabolism-AhR pathway by modulating the gut microbiota, reducing intestinal inflammation, improving intestinal barrier function, enhancing anti-tumor immunity, and effectively inhibiting CAC growth.

Deciphering the molecular and clinical characteristics of TREM2, HCST, and TYROBP in cancer immunity: A comprehensive pan-cancer study.

Background: Hematopoietic cell signal transducer (HCST) and tyrosine kinase-binding protein (TYROBP) are triggering receptors expressed on myeloid cells 2 (TREM2), which are pivotal in the immune response to disease. Despite growing evidence underscoring the significance of TREM2, HCST, and TYROBP in certain forms of tumorigenesis, a comprehensive pan-cancer analysis of these proteins is lacking. Methods: Multiple databases were synthesized to investigate the relationship between TREM2, HCST, TYROBP, and various cancer types. These include prognosis, methylation, regulation by long non-coding RNAs and transcription factors, immune signatures, pathway activity, microsatellite instability (MSI), tumor mutational burden (TMB), single-cell transcriptome profiling, and drug sensitivity. Results: TREM2, HCST, and TYROBP displayed extensive somatic changes across numerous tumors, and their mRNA expression and methylation levels influenced patient outcomes across multiple cancer types. long non-coding RNA (lncRNA) -messenger RNA (mRNA) and TF-mRNA regulatory networks involving TREM2, HCST, and TYROBP were identified, with lncRNA MEG3 and the transcription factor SIP1 emerging as potential key regulators. Further immune analyses indicated that TREM2, HCST, and TYROBP play critical roles in immune-related pathways and macrophage differentiation, and may be significantly associated with TGF-ß and SMAD9. Furthermore, the expression of TREM2, HCST, and TYROBP correlated with the immunotherapy markers TMB and MSI, and influenced sensitivity to immune-targeted drugs, thereby indicating their potential as predictors of immunotherapy outcomes. Conclusion: This study offers valuable insights into the roles of TREM2, HCST, and TYROBP in tumor immunotherapy, suggesting their potential as prognostic markers and therapeutic targets for various cancers.

Xihuang pills induce apoptosis in hepatocellular carcinoma by suppressing phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin pathway.

BACKGROUND: The phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin (PI3K/Akt/mTOR) signalling pathway is crucial for cell survival, differentiation, apoptosis and metabolism. Xihuang pills (XHP) are a traditional Chinese preparation with antitumour properties. They inhibit the growth of breast cancer, glioma, and other tumours by regulating the PI3K/Akt/mTOR signalling pathway. However, the effects and mechanisms of action of XHP in hepatocellular carcinoma (HCC) remain unclear. Regulation of the PI3K/Akt/mTOR signalling pathway effectively inhibits the progression of HCC. However, no study has focused on the XHP-associated PI3K/Akt/mTOR signalling pathway. Therefore, we hypothesized that XHP might play a role in inhibiting HCC through the PI3K/Akt/mTOR signalling pathway. AIM: To confirm the effect of XHP on HCC and the possible mechanisms involved. METHODS: The chemical constituents and active components of XHP were analysed using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS). Cell-based experiments and in vivo xenograft tumour experiments were utilized to evaluate the effect of XHP on HCC tumorigenesis. First, SMMC-7721 cells were incubated with different concentrations of XHP (0, 0.3125, 0.625, 1.25, and 2.5 mg/mL) for 12 h, 24 h and 48 h. Cell viability was assessed using the CCK-8 assay, followed by an assessment of cell migration using a wound healing assay. Second, the effect of XHP on the apoptosis of SMMC-7721 cells was evaluated. SMMC-7721 cells were stained with fluorescein isothiocyanate and annexin V/propidium iodide. The number of apoptotic cells and cell cycle distribution were measured using flow cytometry. The cleaved protein and mRNA expression levels of caspase-3 and caspase-9 were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively. Third, Western blotting and RT-qPCR were performed to confirm the effects of XHP on the protein and mRNA expression of components of the PI3K/Akt/mTOR signalling pathway. Finally, the effects of XHP on the tumorigenesis of subcutaneous hepatocellular tumours in nude mice were assessed. RESULTS: The following 12 compounds were identified in XHP using high-resolution mass spectrometry: Valine, 4-gingerol, myrrhone, ricinoleic acid, glycocholic acid, curzerenone, 11-keto-ß-boswellic acid, oleic acid, germacrone, 3-acetyl-9,11-dehydro-ß-boswellic acid, 5ß-androstane-3,17-dione, and 3-acetyl-11-keto-ß-boswellic acid. The cell viability assay results showed that treatment with 0.625 mg/mL XHP extract decreased HCC cell viability after 12 h, and the effects were dose- and time-dependent. The results of the cell scratch assay showed that the migration of HCC cells was significantly inhibited in a time-dependent manner by the administration of XHP extract (0.625 mg/mL). Moreover, XHP significantly inhibited cell migration and resulted in cell cycle arrest and apoptosis. Furthermore, XHP downregulated the PI3K/Akt/mTOR signalling pathway, which activated apoptosis executioner proteins (e.g., caspase-9 and caspase-3). The inhibitory effects of XHP on HCC cell growth were determined in vivo by analysing the tumour xenograft volumes and weights. CONCLUSION: XHP inhibited HCC cell growth and migration by stimulating apoptosis via the downregulation of the PI3K/Akt/mTOR signalling pathway, followed by the activation of caspase-9 and caspase-3. Our findings clarified that the antitumour effects of XHP on HCC cells are mediated by the PI3K/Akt/mTOR signalling pathway, revealing that XHP may be a potential complementary therapy for HCC.

Frankincense myrrh attenuates hepatocellular carcinoma by regulating tumor blood vessel development through multiple epidermal growth factor receptor-mediated signaling pathways.

BACKGROUND: In traditional Chinese medicine (TCM), frankincense and myrrh are the main components of the antitumor drug Xihuang Pill. These compounds show anticancer activity in other biological systems. However, whether frankincense and/or myrrh can inhibit the occurrence of hepatocellular carcinoma (HCC) is unknown, and the potential molecular mechanism(s) has not yet been determined. AIM: To predict and determine latent anti-HCC therapeutic targets and molecular mechanisms of frankincense and myrrh in vivo. METHODS: In the present study, which was based on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (http://tcmspw.com/tcmsp.php), Universal Protein database (http://www.uniprot.org), GeneCards: The Human Gene Database (http://www.genecards.org/) and Comparative Toxicogenomics Database (http://www.ctdbase.org/), the efficacy of and mechanism by which frankincense and myrrh act as anti-HCC compounds were predicted. The core prediction targets were screened by molecular docking. In vivo, SMMC-7721 human liver cancer cells were transplanted as xenografts into nude mice to establish a subcutaneous tumor model, and two doses of frankincense plus myrrh or one dose of an EGFR inhibitor was administered to these mice continuously for 14 d. The tumors were collected and evaluated: the tumor volume and growth rate were gauged to evaluate tumor growth; hematoxylin-eosin staining was performed to estimate histopathological changes; immunofluorescence (IF) was performed to detect the expression of CD31, α-SMA and collagen IV; transmission electron microscopy (TEM) was conducted to observe the morphological structure of vascular cells; enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of secreted HIF-1α and TNF-α; reverse transcription-polymerase chain reaction (RT-qPCR) was performed to measure the mRNA expression of HIF-1α, TNF-α, VEGF and MMP-9; and Western blot (WB) was performed to determine the levels of proteins expressed in the EGFR-mediated PI3K/Akt and MAPK signaling pathways. RESULTS: The results of the network pharmacology analysis showed that there were 35 active components in the frankincense and myrrh extracts targeting 151 key targets. The molecular docking analysis showed that both boswellic acid and stigmasterol showed strong affinity for the targets, with the greatest affinity for EGFR. Frankincense and myrrh treatment may play a role in the treatment of HCC by regulating hypoxia responses and vascular system-related pathological processes, such as cytokine-receptor binding, and pathways, such as those involving serine/threonine protein kinase complexes and MAPK, HIF-1 and ErbB signaling cascades. The animal experiment results were verified. First, we found that, through frankincense and/or myrrh treatment, the volume of subcutaneously transplanted HCC tumors was significantly reduced, and the pathological morphology was attenuated. Then, IF and TEM showed that frankincense and/or myrrh treatment reduced CD31 and collagen IV expression, increased the coverage of perivascular cells, tightened the connection between cells, and improved the shape of blood vessels. In addition, ELISA, RT-qPCR and WB analyses showed that frankincense and/or myrrh treatment inhibited the levels of hypoxia-inducible factors, inflammatory factors and angiogenesis-related factors, namely, HIF-1α, TNF-α, VEGF and MMP-9. Furthermore, mechanistic experiments illustrated that the effect of frankincense plus myrrh treatment was similar to that of an EGFR inhibitor with regard to controlling EGFR activation, thereby inhibiting the phosphorylation activity of its downstream targets: the PI3K/Akt and MAPK (ERK, p38 and JNK) pathways. CONCLUSION: In summary, frankincense and myrrh treatment targets tumor blood vessels to exert anti-HCC effects via EGFR-activated PI3K/Akt and MAPK signaling pathways, highlighting the potential of this dual TCM compound as an anti-HCC candidate.

Construction of a circRNA-miRNA-mRNA network revealed the potential mechanism of Buyang Huanwu Decoction in the treatment of cerebral ischemia.

BACKGROUND AND AIM: Buyang Huanwu Decoction (BHD) is a traditional Chinese herbal medicine that is effective for treating cerebral ischemia (CI). However, the molecular mechanisms of BHD in CI have not been fully elucidated. In this study, we integrated the circular RNA (circRNA)-microRNA (miRNA)-messenger RNA (mRNA) network of middle cerebral artery occlusion (MACO) rats treated with BHD. METHODS: SD rats were randomly divided into a control group, model group, model+BHD group (2.5, 5, 10 g/kg) and model+butylphthalide (NBP) group (54 mg/kg). The neurological functions of the rats were evaluated by a modified neurological severity scoring (mNSS) system. Pathological lesions were assessed by Nissl staining, and the effects of BHD on neurovascular unit (NVU) associated protein microtubule-associated protein 2 (MAP2), glial fibrillary acidic protein (GFAP) and von Willebrand factor (VWF) were assessed by immunohistochemistry. CeRNA and miRNA microarrays were used to establish the circRNA, miRNA, and mRNA profiles. Finally, a circRNA-miRNA-mRNA ternary transcription network was constructed. RESULTS: BHD improved the neurobehavioral test scores (P < 0.01) and the histopathological changes in ischemic brain tissue in MCAO rats. The expression of MAP2 and VWF decreased and the expression of GFAP increased in the ischemic side brain tissue of MCAO rats (P < 0.01), and treatment with BHD reversed the above changes (P < 0.01 or 0.05). We identified seven, three, and 86 significantly dysregulated circRNAs, miRNAs, and mRNAs, respectively, that were associated with the neuroprotective effects of BHD. Furthermore, bioinformatics analysis showed that these targets may exert therapeutic effects through multiple pathways, such as the VEGF and Hippo signaling pathways. Finally, we constructed a circRNA-miRNA-mRNA network. CONCLUSIONS: In brief, our study provides novel insights into ceRNA-mediated gene regulation in the progression of NVU after CI and the mechanism of action for BHD.

Curcumin inhibits the growth of liver cancer by impairing myeloid-derived suppressor cells in murine tumor tissues.

Curcumin, one of the active ingredients of Curcuma longa (Jianghuang), has been reported to exert multiple bioactivities, including pro-apoptotic and anti-inflammatory activities. In recent years, curcumin has been extensively studied, and it has been revealed that curcumin inhibits the growth of numerous types of cancer. However, to the best of our knowledge, the inhibitory effects of curcumin on the activation or expansion of myeloid-derived suppressor cells (MDSCs) in liver cancer and the underlying mechanism have not yet been determined. Therefore, the present study aimed to investigate the inhibitory effect of curcumin on MDSC activity and the associated anti-neoplastic mechanism in a HepG2 ×enograft mouse model. The effect of curcumin on the viability of Huh-7, MHCC-97H and HepG2 cells in vitro was analyzed using a Cell Counting Kit-8 assay. The effects of curcumin on tumor growth, numbers of MDSCs, expression levels of proteins involved in the toll-like receptor 4 (TLR4)/NF-κB signaling pathway, levels of related inflammatory factors and angiogenesis were determined in HepG2 ×enograft model mice, which were given different doses of curcumin via intragastrical administration. The results of the present study revealed that curcumin inhibited the viability of Huh-7, MHCC-97H and HepG2 cells and the growth of HepG2 ×enograft tumors in mice. Flow cytometric analysis indicated that curcumin reduced the number of MDSCs in mouse xenograft tumors. In addition, the results demonstrated that curcumin inhibited the TLR4/NF-κB signaling pathway and the expression of inflammatory factors, including IL-6, IL-1ß, prostaglandin E2 and cyclooxygenase-2, in mouse xenograft tumors. Furthermore, curcumin suppressed the secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte-colony stimulating factor (G-CSF), which are essential factors for MDSCs modulation, in tumor tissues. Additionally, curcumin was revealed to inhibit angiogenesis, which was demonstrated by the downregulation of the expression levels of vascular endothelial growth factor, CD31 and α-smooth muscle actin in western blotting, immunohistochemistry and immunofluorescence experiments. In conclusion, the findings of the present study identified a novel mechanism via which curcumin may suppress the growth of liver cancer by reducing the numbers of MDSCs and subsequently disrupting the process of angiogenesis. These conclusions were supported by the observed inactivation of the TLR4/NF-κB signaling pathway-mediated inflammatory response and the downregulation of GM-CSF and G-CSF secretion in xenograft tissues.

Author Correction: Directed self-assembly of herbal small molecules into sustained release hydrogels for treating neural inflammation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Rhein Suppresses Neuroinflammation via Multiple Signaling Pathways in LPS-Stimulated BV2 Microglia Cells.

As a bioactive absorbed compound of rhubarb, Rhein is applied for the treatment of brain injury. However, the underlying pharmacological mechanisms remain unclear. In this study, we aimed to explore antineuroinflammatory functions and underlying mechanisms of Rhein in vitro. BV2 microglia cells were chosen and irritated by LPS. The influence of Rhein on cell viability was determined using MTT assay. We finely gauged the proinflammatory cytokines of TNF-α and IL-1ß through tests of immunofluorescence staining, ELISA, RT-qPCR, and western blot. Additionally, mediators including IL-6, IL-12, iNOS, and IL-10 were surveyed by ELISA. Furthermore, protein levels of the underlying signaling pathways (PI3K/Akt, p38, ERK1/2, and TLR4/NF-κB) were tested adopting western blot. We found that Rhein reduced the secretion of pivotal indicators including TNF-α and IL-1ß, effectively restraining their mRNA and protein expression in LPS-activated BV2 microglial cells. Besides, Rhein treatment demoted the production of IL-6, IL-12, and iNOS and promoted the excretion of IL-10. Subsequent mechanistic experiments revealed that Rhein obviously downregulated the phosphorylation levels of PI3K, Akt, p38, and ERK1/2 and simultaneously upregulated the PTEN expression. In addition, Rhein antagonized the increase of TLR4, p-IκBα, and NF-κB. In summary, Rhein suppresses neuroinflammation via multiple signaling pathways (PI3K/Akt, p38, ERK1/2, and TLR4/NF-κB) in LPS-stimulated BV2 microglia cells. This study highlights a natural agent for prevention and treatment of neuroinflammation.

Chitosan Hydrogel Delivery System Containing Herbal Compound Functions as a Potential Antineuroinflammatory Agent.

Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is an anthraquinone compound mainly isolated from the herbal medicine rhubarb. It possesses a wide spectrum of pharmacological effects. However, the lack of sustained release properties and the poor bioavailability hinder clinical transformation. Hydrogel-based drug delivery system provides an ideal carrier to improve the release control and the therapeutic efficacy of drugs. Herein, we present a chitosan hydrogel for the delivery of rhein. This rhein-chitosan hydrogel (CS-Rh gel) exhibited superior characteristics including mechanical strength, sustained release, and low toxicity. For medical application, the enzyme-linked immunosorbent assay and Western blot analyses indicated that CS-Rh gel significantly suppressed the production of proinflammatory cytokines including TNF-α and IL-1ß in lipopolysaccharide-induced BV2 cells. Additionally, CS-Rh gel blocked the neuroinflammation-related mitogen-activated protein kinase (JNK, ERK, and p38)-signaling pathways. Interestingly, these inhibitory effects at 48 h outperformed the pharmacologic actions at 24 h, showing that the CS-Rh gel exerted optimal sustained antineuroinflammation. This study highlights a novel chitosan hydrogel containing rhein used as a potential antineuroinflammatory agent.

Directed self-assembly of herbal small molecules into sustained release hydrogels for treating neural inflammation.

Self-assembling natural drug hydrogels formed without structural modification and able to act as carriers are of interest for biomedical applications. A lack of knowledge about natural drug gels limits there current application. Here, we report on rhein, a herbal natural product, which is directly self-assembled into hydrogels through noncovalent interactions. This hydrogel shows excellent stability, sustained release and reversible stimuli-responses. The hydrogel consists of a three-dimensional nanofiber network that prevents premature degradation. Moreover, it easily enters cells and binds to toll-like receptor 4. This enables rhein hydrogels to significantly dephosphorylate IκBα, inhibiting the nuclear translocation of p65 at the NFκB signalling pathway in lipopolysaccharide-induced BV2 microglia. Subsequently, rhein hydrogels alleviate neuroinflammation with a long-lasting effect and little cytotoxicity compared to the equivalent free-drug in vitro. This study highlights a direct self-assembly hydrogel from natural small molecule as a promising neuroinflammatory therapy.

Plasma Metabolomics Analysis Based on GC-MS in Infertile Males with Kidney-Yang Deficiency Syndrome.

INTRODUCTION: Chinese medicine syndrome diagnosis is the key requisite in the treatment of male infertility with traditional Chinese medicine (TCM). Kidney-Yang deficiency syndrome (KYDS) is the critical Chinese medicine syndrome of male infertility. To explore the modernized mechanisms of KYDS in male infertility, this study aims to investigate the metabolomics of males with KYDS. METHODS: The gas chromatography-mass spectrometry method was applied to analyze the plasma samples of 67 infertile males with KYDS compared with 55 age-matched healthy controls. The chemometric methods including principal component and partial least squares-discriminate analyses were employed to identify the potential biochemical patterns. With the help of the variable importance for the projection and receiver operating characteristic curve analyses, the potential biomarkers were extracted to define the clinical utility. Simultaneously the high-quality KEGG metabolic pathways database was used to identify the related metabolic pathways. RESULTS: The metabolomics profiles of infertile males with KYDS including 10 potential biomarkers and six metabolic pathways were identified. They precisely distinguished infertile males with KYDS from healthy controls. CONCLUSIONS: These potential biomarkers and pathways suggest the substantial basis of infertile males with KYDS. The metabolomics profiles highlight the modernized mechanisms of infertile males with KYDS.

Nine traditional Chinese herbal formulas for the treatment of depression: an ethnopharmacology, phytochemistry, and pharmacology review.

Depression is a major mental disorder, and is currently recognized as the second-leading cause of disability worldwide. However, the therapeutic effect of antidepressants remains unsatisfactory. For centuries, Chinese herbal formulas (CHFs) have been widely used in the treatment of depression, achieving better therapeutic effects than placebo and having fewer side effects than conventional antidepressants. Here, we review the ethnopharmacology, phytochemistry, and pharmacology studies of nine common CHFs: "banxia houpo" decoction, "chaihu shugansan", "ganmaidazao" decoction, "kaixinsan", "shuganjieyu" capsules, "sinisan", "wuling" capsules, "xiaoyaosan", and "yueju". Eight clinical trials and seven meta-analyses have supported the theory that CHFs are effective treatments for depression, decreasing Hamilton Depression Scale scores and showing few adverse effects. Evidence from 75 preclinical studies has also elucidated the multitarget and multipathway mechanisms underlying the antidepressant effect of the nine CHFs. Decoctions, capsules, and pills all showed antidepressant effects, ranked in descending order of efficacy. According to traditional Chinese medicine theory, these CHFs have flexible compatibility and mainly act by soothing the liver and relieving depression. This review highlights the effective treatment choices and candidate compounds for patients, practitioners, and researchers in the field of traditional Chinese medicine. In summary, the current evidence supports the efficacy of CHFs in the treatment of depression, but additional large-scale randomized controlled clinical trials and sophisticated pharmacology studies should be performed.