Network pharmacology, bioinformatics, and experimental validation to identify the role of Hedyotis diffusa willd against gastric cancer through the activation of the endoplasmic reticulum stress.

Background: Globally, gastric cancer (GC) is recognized as the third leading cause of cancer-related deaths and the fifth most prevalent malignant disease. Multiple studies have indicated that Hedyotis diffusa Willd, in pinyin, called Bai Hua She Cao (BHSSC), a traditional Chinese medicine (TCM) is an herbal remedy for cancer treatment. However, the specific mechanisms underlying its anti-tumor properties and mode of action are still unclear. Methods: To determine the role of BHSSC in GC, candidate target genes were selected from The Encyclopedia of Traditional Chinese Medicine (ETCM) and analyzed using network pharmacology, bioinformatics, and experimental validation. Differentially expressed genes (DEGs) associated with gastric cancer were obtained from RNA sequencing (RNA-seq) data sourced from The Cancer Genome Atlas-Stomach adenocarcinoma (TCGA-STAD). The Reactome Pathway was examined using Analysis Tools, while KEGG pathways were analyzed using KOBAS. Gene Ontology (GO) evaluations were performed using WebGestalt and DAVID. The relationships between proteins were investigated using the STRING database. Furthermore, cell viability, colony formation, and cell migration ability were conducted in gastric cancer cells, BGC-823 and MGC-803. Results: Network pharmacology and bioinformatics analyses revealed a significant association between BHSSC and metabolic pathways. In vitro experiments demonstrated that BHSSC effectively suppressed gastric cancer cell proliferation and colony formation, inhibited cell migration, and activated the endoplasmic reticulum (ER) stress. Furthermore, it was found that enhancement of the expression of IRE1α and BIP is the mechanism by which BHSSC activates ER stress. Conclusions: The findings suggest that BHSSC exerts its effects through modulation of metabolic pathways, leading to the suppression of cell proliferation, inhibition of cell migration, and activation of the endoplasmic reticulum. These results provide valuable insights into the mechanisms underlying the therapeutic effects of BHSSC in GC and support its potential as a novel treatment option.

Molecular Targets and Mechanisms of Hedyotis diffusa Willd. for Esophageal Adenocarcinoma Treatment Based on Network Pharmacology and Weighted Gene Co-expression Network Analysis.

BACKGROUND: Hedyotis diffusa Willd. (HDW) is a common anticancer herbal medicine in China, and its therapeutic effectiveness has been demonstrated in a range of cancer patients. There is no consensus about the therapeutic targets and molecular mechanisms of HDW, which contains many active ingredients. AIM: To clarify the mechanism of HDW for esophageal adenocarcinoma (EAC), we utilized network pharmacology and weighted gene co-expression network analysis methods (WGCNA). METHODS: The gene modules that were linked with the clinical features of EAC were obtained through the WGCNA method. Then, the potential target genes were retrieved through the network pharmacology method in order to determine the targets of the active components. After enrichment analysis, a variety of signaling pathways with significant ratios of target genes were found, including regulation of trans-synaptic signaling, neuroactive ligand-receptor interaction and modulation of chemical synaptic transmission. By means of protein-protein interaction (PPI) network analysis, we have successfully identified the hub genes, which were AR, CNR1, GRIK1, MAPK10, MAPT, PGR and PIK3R1. RESULT: Our study employed molecular docking simulations to evaluate the binding affinity of the active components with the hub gene. The identified active anticancer constituents in HDW are scopoletol, quercetin, ferulic acid, coumarin, and trans-4-methoxycinnamyl alcohol. CONCLUSION: Our findings shed light on the molecular underpinnings of HDW in the treatment of EAC and hold great promise for the identification of potential HDW compounds and biomarkers for EAC therapy.

Toxicity study of compound granules of Hedyotis diffusa: Acute toxicity and long-term toxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: The clinical efficacy of the hospital preparation compound granules of Hedyotis diffusa (CGHD), which is composed of Hedyotis diffusa Willd, Smilax china L., Solanum lyratum Thunb., has accumulated a good reputation over the past decades. However, because it is a hospital preparation, few researchers have paid attention to it, resulting in a lack of systematic basic research studies. Thus, it is not clear whether there are safety concerns that restrict its clinical application, and toxicological evaluation of CGHD is needed. AIM OF THE STUDY: The aim of this study was to evaluate the safety of CGHD by conducting acute toxicity and long-term toxicity experiments, with the objective of providing evidence for its clinical safety and a theoretical foundation for its clinical application. MATERIALS AND METHODS: KM mice were selected for the acute toxicity experiment and were administered water or CGHD-E 3 times within 24 h. The reactions of the animals to CGHD treatment were observed and recorded within 1 h after administration and then once a day for 14 consecutive days. SD rats were selected to conduct the long-term toxicity experiment. The drug-treated groups were administered different doses of CGHD-E, which were equivalent to 10 times, 20 times and 50 times the clinical dose in humans. The rats were administered the drug for 28 consecutive days. After 28 days, the animals were sacrificed, and routine blood tests, blood coagulation function analysis, liver and kidney function tests, and glycolipid metabolism related tests were conducted. The major organs of the rats were collected to calculate organ coefficients and perform hematoxylin-eosin (HE) staining. RESULTS: In the CGHD-E acute toxicity experiment, the drug-treated groups did not show adverse reactions or poisoning symptoms, and the maximum tolerated dose of CGHD-E in mice was greater than 45.072 g/kg. In the long-term toxicity experiment, drug-treated rats generally exhibited a good condition, but continuous administration decreased on body weight and food intake, especially in male rats. Coagulation function alterations and the impact on the liver during long-term drug administration were also assessed, which should be emphasized in clinical applications. No significant toxic effects were observed according to routine blood tests or test of liver and kidney function, glucose and lipid metabolism, or ion metabolism. CONCLUSIONS: The results of this study showed that CGHD was nontoxic or had low toxicity, providing not only a scientific basis for its clinical application, determining the appropriate clinical dose and monitoring clinical toxicity but also theoretical support for subsequent clinical drug trials.

Evaluation of therapeutic mechanism of Hedyotis diffusa Willd (HDW)‒Scutellaria barbata (SB) in clear cell renal cell carcinoma via single-cell RNA sequencing and network pharmacology.

OBJECTIVE: The present study aimed to investigate the therapeutic mechanism of Hedyotis diffusa Willd (HDW) and Scutellaria barbata (SB) in ccRCC using a combination of single-cell RNA sequencing (scRNA-seq) and network pharmacology. METHODS: The active ingredients and potential molecular targets of HDW-SB were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform. Gene expression data (GSE53757) were obtained from the Gene Expression Omnibus database. The hub genes of HDW-SB against ccRCC were identified via the protein-protein interaction network, and further analyzed by molecular complex detection. The roles of these genes in the diagnosis and immune infiltration of ccRCC were analyzed. The clinical significance of hub genes was verified using scRNA-seq data (GSE121638) and molecular docking. RESULTS: Following the PPI network analysis, 29 hub genes of HDW-SB against ccRCC were identified. All hub genes, except for CENPE, had significantly different expressions in tumor tissue and a more accurate diagnosis of ccRCC. Fifteen cell clusters were defined based on the scRNA-seq dataset, and the clusters were annotated as six cell types using marker genes. TYMS and KIAA0101 from hub genes were highly expressed in NK cells. Three active compounds, quercetin, luteolin, and baicalein, were found to target TYMS and KIAA0101 from the compound-target interaction network. CONCLUSION: 29 hub genes of HDW-SB against ccRCC were identified and showed good performance in terms of diagnosis and prognosis. Moreover, among these hub genes docking with the main ingredients of HDW-SB, TYMS and KIAA0101 exerted anti-ccRCC effects through NK cells.

The anti-inflammatory effects of Hedyotis diffusa Willd on SLE with STAT3 as a key target.

ETHNOPHARMACOLOGICAL RELEVANCE: Hedyotis diffusa Willd, also named Scleromitrion diffusum (Willd.) R.J. Wang, is one medical herb, which has been traditionally used by the She nationality in China. And H. diffusa represents a beneficial effect on Systemic lupus erythematosus (SLE) treatment in clinic. AIM OF THE STUDY: The underlying mechanisms of the protective effects of H. diffusa on SLE remain unclear. In this study, we treated MRL/lpr mice with H. diffusa water extract (HDW) to assess its therapeutic effects and verified its regulating signalling pathway through cytological experiments. MATERIALS AND METHODS: In the present study, the constituents of HDW were analysed through ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and SCIEX OS software. The protective activity and underlying mechanisms were studied in a MRL/lpr lupus mouse model. The blood cells, autoantibodies, metabolites and the cytokines in serum were identified with a hematology analyzer, specific ELISA kit, GC/MS system and cytometric assays. The histological and immunohistochemical analysis were engaged in the morphologic, and the expression and translocation of the crucial protein observation. The dual luciferase reporter assay was applied to identifying the regulative activity of HDW. The transcription and translation expression of the protein was studied by real-time PCR and Western blot assays. The network pharmacology analysis was employed to predict the IL-6/STAT3 pathway regulators and the screen the STAT3 inhibitors in HDW. RESULTS: The results revealed the capability of HDW to attenuate the production of autoantibodies, secretion of inflammatory cytokines (IL-6 and IFN-γ), and suppressed the IgG and C3 deposition, the development of glomerular lesions in MRL/lpr mice. Serum metabolomics study showed the improvement in serum metabolites, especially aminoacyl-tRNA biosynthesis, by HDW. IL-6 was clarified to be highly associated with the significantly changed metabolites in network analysis. We further demonstrated the effects of HDW on the IL-6/STAT3 pathway in vivo and in vitro. CONCLUSIONS: This study suggested that HDW exerts a therapeutic effect in SLE model mice by suppressing the IL-6/STAT3 pathway.

The protective capability of Hedyotis diffusa Willd on lupus nephritis by attenuating the IL-17 expression in MRL/lpr mice.

Lupus nephritis (LN), the most severe organ manifestation of systemic lupus erythematosus (SLE), is generally treated with glucocorticoids (GC) in clinical practice, leading to drug resistance and adverse effects in the long term. Fortunately, the combination of GC and traditional Chinese medical prescriptions can attenuate the adverse effects and improve therapeutic efficiency. Hedyotis diffusa Willd (HDW) is one of the most commonly used herbal compounds for LN treatment, which exhibits "heat-clearing" and "detoxification" effects. However, the underlying pharmacological mechanism remains unclear. The present study identified the chemical compounds in HDW extract with UPLC-Q-TOF-MS/MS. A total of 49 components were identified in the HDW extract, and the IL-17 signaling pathway was highly enriched by network pharmacological analysis. MRL/lpr model mice, reflecting the spontaneous development of LN, were used to evaluate the protective activity and investigate the underlying mechanism of the combination treatment. The white blood cell content (WBC), including lymphocytes and neutrophils, cytokines (IL-6, MCP-1, TNF-a), and various autoantibodies (ANA, ab-dsDNA, ab-snRNP/sm) in the blood of MRL/lpr mice were significantly improved by the intragastric administration of HDW. Additionally, the expression of STAT3, IL-17, Ly6G, and MPO in the kidney and neutrophil NETosis were ameliorated with HDW treatment. The pathological and morphological analysis suggested that HDW application could reduce urinary protein levels and inflammatory cell infiltration and inhibit glomerular interstitial cell proliferation. Hence, HDW might ameliorate lupus nephritis by inhibiting IL-6 secretion and STAT3-induced IL-17 expression. The active compounds in HDW were predictively selected with computational methods. The docking affinity of asiatic acid, neoandrographolide to IL-6, glycyrrhetinic acid, oleanolic acid, ursolic acid, and wilforlide A to STAT3 are extremely high. In conclusion, the IL-6 and STAT3/IL-17signaling pathways could be critical regulative targets of HDW on LN.

Protective Effect of Hedyotis diffusa Willd. Ethanol Extract on Isoniazid-Induced Liver Injury in the Zebrafish Model.

Objective: This study aims to investigate the hepatoprotective effect and molecular mechanism of Hedyotis diffusa Willd. ethanol extract (HDWE) against isoniazid (INH)-induced liver injury in the zebrafish model. Methods: INH-induced liver injury model was established by adding an immersion bath of INH in 3 days post-fertilisation (dpf) healthy transgenic zebrafish with liver-specific fluorescence (L-FABP: EGFP). HDWE and INH were given to the zebrafish to observe liver morphology and pathology, fluorescence intensity, and the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and superoxide dismutase (SOD), as well as the content of glutathione (GSH). The chemical composition of HDWE was analysed using high-performance liquid chromatography coupled with a quadrupole-time-of flight hybrid mass spectrometer (HPLC-Q-TOF-MS). The bioactive compounds, molecular targets and signalling pathways of HDWE were predicted using network pharmacology. Subsequently, molecular docking was adopted to analyze the affinities between the bioactive components and targets by Autodock. Finally, in vitro experiments were conducted to further verify the findings. Results: Our findings showed that HDWE had a remarkable protective effect on INH-induced liver injury in zebrafish. Twenty compounds in HDWE were identified. Nineteen hub targets were identified as possible targets of HDWE, and a compound-target-pathway network was constructed. Nine bioactive compounds, ten molecular targets, and seven key signalling pathways were found to play a pivotal role in the hepatoprotective effect of HDWE against INH-induced liver injury. In vitro studies revealed that the important bioactive compound quercetin-3-O-sambubioside (QSA) could significantly reverse INH-induced cell viability decreases and had a significant effect on the associated targets predicted by network pharmacology and molecular docking. Conclusion: In this study, through the research of hepatoprotective effect of HDWE and bioinformatics analysis, the bioactive compounds, important pathways and key molecular targets were discovered. These findings could provide scientific evidence for the use of HDW in liver injury and prove to help explore its efficacy and the mechanism of action.

Ursolic acid silences CYP19A1/aromatase to suppress gastric cancer growth.

INTRODUCTION: Gastric cancer (GCa) is a malignancy with few effective treatments. Ursolic acid (UA), a bioactive triterpenoid enriched in Hedyotis diffusa Willd, known to suppress GCa without identified target. CYP19A1 (cytochrome P450 family 19A1; also known as aromatase, Ar) was correlated to GCa prognosis. Relatedly, Ar silencers, which halt the expression of Ar exhibited anti-GCa effects in experimental models, are currently being investigated. METHOD: The docking simulation score of UA was compared with Ar inhibitors, e.g., letrozole, exemestane, in Ar protein crystallization. Hedyotis diffusa Willd ethanol extract, UA, or 5-fluracil were applied onto AGS, SC-M1, MKN45 GCa cells for cancer inhibition tests. Immunoblot for measuring gene expressions upon drug treatments, or gene knockdown/overexpression. Treatments were also applied in a MKN45 implantation tumor model. A web-based GCa cohort for Ar expression association with prognosis was performed. RESULT: The ethanol extracts of Hedyotis diffusa Willd, enrich with UA, exhibited cytotoxic activity against GCa cells. Molecular docking simulations with the 3D Ar structure revealed an excellent fitting score for UA. UA increase cytotoxic, and suppressed colony, in addition to its Ar silencing capacity. Moreover, UA synergistically facilitated 5-FU, (a standard GCa treatment) regimen in vitro. Consistent with those results, adding estradiol did not reverse the cancer-suppressing effects of UA, which confirmed UA acts as an Ar silencer. Furthermore, UA exhibited tumor-suppressing index (TSI) score of 90% over a 6-week treatment term when used for single dosing in xenograft tumor model. In the clinical setting, Ar expression was found to be higher in GCa tumors than normal parental tissue from the TCGA (The Cancer Genome Atlas) cohort, while high Ar expression associated with poor prognosis. Together, the results indicate UA could be used to treat GCa by silencing Ar expression in GCa. Hedyotis diffusa Willd ethanol extract could be an functional food supplements.

Imperatae rhizoma-Hedyotis diffusa Willd. herbal pair alleviates nephrotic syndrome by integrating anti-inflammatory and hypolipidaemic effects.

BACKGROUND: Nephrotic syndrome (NS) is a common nephropathy with a complex and diverse aetiology. Both Imperatae rhizoma and Hedyotis diffusa Willd. are herbs that are widely used as medicine and functional food. In traditional Chinese medicine theory, they are used as an herbal pair (HP) to treat inflammation-related diseases in the clinic, especially disorders of the kidney. PURPOSE: This study aimed to investigate the anti-inflammatory and hypolipidaemic effects of HP in an NS rat model and provide scientific data for its clinical application. METHODS: An NS model was established by two-dose injection of Sprague-Dawley rats with adriamycin. Seven groups, including the sham, model, HP treatment (0.25, 0.5 and 1.0 g/kg/d), prednisone (positive control, 5 mg/kg/d), and atorvastatin (positive control, 4 mg/kg/d) groups, were tested. The biochemical indexes of renal function and inflammatory cytokines were determined by ELISA kits and/or qPCR assays, and the crucial protein involved in the signalling pathway were subsequently tested by qPCR and/or Western blotting. Based on specific compounds identified by LC-Q-TOF-MS, network pharmacological study was carried out. RESULTS: The levels of BUN, Scr, Upro, UA, Alb, TC, TG, and LDL-C were significantly elevated in model rats. HP treatment for four weeks improved the renal function and the dyslipidaemia by decreasing the levels of all parameters, except BUN and Scr. HP treatment (0.5 and 1.0 g/kg/d) upregulated the expression of PPARγ, CYP7b1, and LDLR in the liver, while it down-regulated PCSK9, showing a regulatory effect on lipid metabolism disorder. The levels of TNF-α and IL-1ß in the plasma and the mRNA expression of TNF-α, IL-1ß, MCP-1, and TGF-ß1 in the kidney were decreased in HP groups, revealing its anti-inflammatory effect in NS rats. The HP exerted an alleviation effect on the inflammatory response through the NF-κB pathway by inhibiting the mRNA and protein expression of p50 and p65. There were 34 compounds identified or tentatively characterized in HP. In the network pharmacological study, PPARG(PPARγ), PCSK9, RELA(p65), and NF-κB1(p50) were the top 20 targets for HP, supporting the animal experimental results. CONCLUSION: HP exhibited protective effects on NS rats. These effects might be closely related to the inhibition of NF-κB and PCSK9-LDLR and activation of the PPARγ-CYP7B1 signalling pathways.

Exploring the mechanisms of anti-ovarian cancer of Hedyotis diffusa Willd and Scutellaria barbata D. Don through focal adhesion pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Hedyotis diffusa Willd and Scutellaria barbata D.Don (HD-SB) pairing were widely used as traditional medicine known for their anti-tumor effects. However, the inhibitory effect of HD-SB on ovarian cancer and its potential mechanisms were still not clear. AIM OF THE STUDY: Our study identified the anti-tumor effect of HD-SB on ovarian cancer and analyzed the potential mechanisms by the network pharmacology and molecular docking method. MATERIALS AND METHODS: The inhibitory effect of HD-SB combination on the growth and migration of ovarian cancer was detected by MTT and transwell assays. The effective ingredients of HD-SB and their potential targets were obtained from the Traditional Chinese Medicines for Systems Pharmacology Database (TCMSP), the GeneCards database, and the UniProt database. The relationships between active ingredients of HD-SB and potential targets or pathways of ovarian cancer were analyzed by String database, Cytoscape 3.7.2 software, and David 6.7 online database. The anti-ovarian cancer targets of HD-SB in the focal adhesion pathway were identified by RT-qPCR and molecular docking. RESULTS: HD-SB combination significantly inhibited the proliferation and migration of ovarian cancer cells. We observed that the 1:2 ratio of HD-SB had the lowest IC50 value. 60 gene targets of 33 active ingredients in HD-SB were selected by pharmacokinetic parameters. The network pharmacological analysis showed that quercetin, luteolin, and baicalein might be the important anti-ovarian cancer ingredients in HD-SB, and the inhibitory effects of these three ingredients on the proliferation of ovarian cancer cells were verified respectively. Functional enrichment results suggested that HD-SB inhibited ovarian cancer growth and migration mainly through the focal adhesion pathway and the potential targets were EGFR, MAPK1, VEGFA, and PIK3CG. CONCLUSIONS: HD-SB pairing significantly inhibited the proliferation and migration of ovarian cancer. Using network pharmacological methods and validation experiments, we found that HD-SB might, at least partially, inhibit ovarian cancer through the focal adhesion pathway. We believed that the HD-SB combination could be a potential therapeutic drug for the treatment of ovarian cancer patients.

The effectiveness of Hedyotis diffusa Willd extract in a mouse model of experimental autoimmune prostatitis.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a frustrating and often debilitating disease. Current studies have shown that Traditional Chinese Medicine (TCM) can improve patients' quality of life and alleviate CP/CPPS symptoms. In this study, the efficacy of Hedyotis diffusa Willd aqueous extraction in experimental autoimmune prostatitis (EAP) mice models was revealed. The C57BL/6 mice were randomly assigned to three groups. Except for the control group, all other groups were subcutaneously injected with 0.2 ml emulsion of T2 peptide, on day 0 and day 14, for inducing EAP models. After the EAP modelling, oral saline was given to the model group, while the H. diffusa group was treated with aqueous extract of H. diffusa Willd. Micturition habits and withdrawal response frequencies were measured. Haematoxylin and eosin staining and immunohistochemistry were used to investigate inflammatory cell infiltration and TNF-α in the prostate tissue respectively. TNF-α levels in the serum were evaluated by ELISA. The H. diffusa Willd aqueous extraction considerably reduced the urine spots number and increased the pain threshold in H. diffusa group. H. diffusa group showed significantly reduced inflammatory lesion and inflammatory cell infiltration than the model group. The levels of TNF-α in H. diffusa group were considerably reduced.

A Potential Herbal Adjuvant Combined With a Peptide-Based Vaccine Acts Against HPV-Related Tumors Through Enhancing Effector and Memory T-Cell Immune Responses.

Viral infection is associated with many types of tumorigenesis, including human papillomavirus (HPV)-induced cervical cancer. The induction of a specific T-cell response against virus-infected cells is desired to develop an efficient therapeutic approach for virus-associated cancer. Chinese herbal medicine (CHM) has a long history in the treatment of cancer patients in Asian countries. Hedyotis diffusa Willd (Bai Hua She She Cao, BHSSC) is frequently used clinically and has been shown to inhibit tumor growth in vitro. However, in vivo data demonstrating the antitumor efficacy of BHSSC are still lacking. We showed that BHSSC induces murine and human antigen-presenting cell (APC) activation via the MAPK signaling pathway and enhances antigen presentation in bone marrow-derived dendritic cells (BMDCs) in vitro. Furthermore, we identified that treatment with BHSSC leads to improved specific effector and memory T-cell responses in vivo. Variant peptide-based vaccines combined with BHSSC improved antitumor activity in preventive, therapeutic, and recurrent HPV-related tumor models. Furthermore, we showed that rutin, one of the ingredients in BHSSC, induces a strong specific immune response against HPV-related tumors in vivo. In summary, we demonstrated that BHSSC extract and its active compound, rutin, can be used as adjuvants in peptide-based vaccines to increase immunogenicity and to bypass the requirement of a conditional adjuvant.

Transcriptional regulation of G2/M regulatory proteins and perturbation of G2/M Cell cycle transition by a traditional Chinese medicine recipe.

ETHNOPHARMACOLOGICAL RELEVANCE: Hedyotis diffusa Willd. (H) and Scutellaria barbata D.Don (S) are ancient anti-cancer Chinese herb medicines. When combined, known as HS, it is one of the most commonly prescribed Chinese Medicines for cancer patients today in China. AIM OF THE STUDY: The prevention of disease progression is a dominant concern for the growing number of men with prostate cancer. The purpose of this work is to evaluate the action and mode of action of Chinese Medicine recipe HS in inhibiting prostate cancer progression in preclinical models. METHODS: Effects of HS were analyzed in prostate cancer cell lines by evaluating proliferation, cell cycle profile, DNA damage and key regulators responsible for G2 to M phase transition. The transcriptional activities of these regulators were determined by RT-PCR and ChIP. The efficacy of HS in vitro was validated in an animal model. RESULTS: HS treatment was observed to reduce DNA content and accumulated prostate cancer cells at the G2/M phase. Immunolabeling for phospho-Histone H3 in association with nocodazole to capture mitotic cells confirmed that HS impeded G2 to M transition. After excluding DNA damage-induced G2 arrest, it was revealed that HS reduced expression of Cyclin B1, CDK1, PLK1 and Aurora A at both protein and mRNA levels, with concomitant reduction of H3K4 tri-methylation at their promoter-regions. Animals that received oral administration of HS with a dosage relevant to clinical application showed reduced tumor volume and weight with a reduction of Cyclin B1, CDK1, PLK1 and Aurora A protein levels. CONCLUSIONS: HS acts by impeding the G2 to M transition of prostate cancer cells. It is likely that the mode of action is transcriptionally suppressing proteins governing mitotic entry, without eliciting significant DNA damage.

Antitumor potential of Hedyotis diffusa Willd: A systematic review of bioactive constituents and underlying molecular mechanisms.

Cancer is a major cause of death in the world. Chemotherapy can extend the life of cancer patients to some extent, but the quality of life is reduced. Therefore, the quest for more efficient and less toxic medication strategies is still at the forefront of current research. Hedyotis diffusa Willd (HDW), a Chinese herb medicine, has received great attention in the past two decades and has been well documented in clinics for antitumor activity in a variety of human cancers. This review discussed a total of 58 different kinds of active antitumor components isolated from HDW, including iridoids, flavonoids, flavonol glycosides, anthraquinones, phenolic acids, and their derivatives, sterols, and volatile oils. Their antitumor activities include inhibition of tumor cell proliferation, induction of tumor cell apoptosis and tumor angiogenesis, regulation of the host immune response, anti-inflammatory and antioxidant, and protective autophagy. Besides, we provide up-to-date and systematic evidence for HDW antitumor activities and the possible underlying molecular mechanisms and reference for further development of novel drugs and dosage formulation in control of human cancers.

Investigating the Multi-Target Pharmacological Mechanism of Hedyotis diffusa Willd Acting on Prostate Cancer: A Network Pharmacology Approach.

Hedyotis diffusa Willd (HDW) is one of the most well-known herbs used in the treatment of prostate cancer. However, the potential mechanisms of its anti-tumor effects have not been fully explored. Here, we applied a network pharmacology approach to explore the potential mechanisms of HDW against prostate cancer (PCa). We obtained 14 active compounds from HDW and 295 potential PCa related targets in total to construct a network, which indicated that quercetin and ursolic acid served as the main ingredients in HDW. Mitogen-activated Protein Kinase 8 (MAPK8), Interleukin 6 (IL6), Vascular Endothelial Growth Factor A (VEGFA), Signal Transducer and Activator of Transcription 3 (STAT3), Jun Proto-Oncogene (JUN), C-X-C Motif Chemokine Ligand 8 (CXCL8), Interleukin-1 Beta (IL1B), Matrix Metalloproteinase-9 (MMP9), C-C Motif Chemokine Ligand 2 (CCL2), RELA Proto-Oncogene (RELA), and CAMP Responsive Element Binding Protein 1 (CREB1) were identified as key targets of HDW in the treatment of PCa. The protein-protein interaction (PPI) cluster demonstrated that CREB1 was the seed in this cluster, indicating that CREB1 plays an important role in connecting other nodes in the PPI network. This enrichment demonstrated that HDW was highly related to translesion synthesis, unfolded protein binding, regulation of mitotic recombination, phosphatidylinositol and its kinase-mediated signaling, nucleotide excision repair, regulation of DNA recombination, and DNA topological change. The enrichment results also showed that the underlying mechanism of HDW against PCa may be due to its coordinated regulation of several cancer-related pathways, such as angiogenesis, cell differentiation, migration, apoptosis, invasion, and proliferation.

Systems pharmacology uncover the mechanism of anti-non-small cell lung cancer for Hedyotis diffusa Willd.

Non-small cell lung cancer (NSCLC) has become one of the most general malignancies in the world and has been shown to be the leading cause of cancer-related deaths. Traditional Chinese medicine (TCM) is considered to be a useful medicine for survival, and has been used in Asia for thousands of years. Hedyotis diffusa Willd (HDW) is an important folk herb that is used in clinical treatment of various cancers in various Chinese medicine prescriptions. However, its underlying mechanism of action remains unclear. Presently, we used an innovative system-pharmacology platform to systematically uncover the pharmacological mechanisms of HDW in the treatment of NSCLC from molecules, targets, and pathway levels. The results show that HDW treatment of NSCLC may activate immunity, achieve anti-inflammatory, anti-proliferative and anti-migration therapeutic effects by regulating multiple pathways. This research provides a new idea for understanding the mechanism of TCM and promotes to develop potential drugs from HDW in modern medicine.

Simultaneous determination of fourteen compounds of Hedyotis diffusa Willd extract in rats by UHPLC-MS/MS method: Application to pharmacokinetics and tissue distribution study.

A rapid, sensitive and selective ultra high-performance liquid chromatography-tandem mass spectrometry UHPLC-MS/MS method has been developed and validated for the simultaneous determination of fourteen bioactive ingredients (gallic acid, geniposidic acid, protocatechuic acid, caffeic acid, ferulic acid, scopoletin, apigenin-7-o-glucuronide, daidzein, apigenin, ursolic acid, oleanolic acid, ß-sitosterol, coniferin, and stigmasterol) in the plasma and tissues of rats. Danshensu and icariin were used as internal standards (IS1 and IS2). The chromatographic separation was achieved by using an Agilent ZORBAX RRHD Eclipse Plus C18 column (2.1 mm × 50 mm, 1.8 µm) with gradient elution using mobile phase, which consisted of 0.1% acetic acid water (solvent A) and methanol (solvent B) and pumped at a flow rate of 0.3 mL/min. Mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode utilizing electrospray ionization (ESI) in positive and negative mode. The plasma samples were pretreated via protein precipitation with 300 µL of methanol containing 0.1% (v/v) formic acid and organ homogenates were processed by solid-phase extraction (SPE) with Waters Oasis HLB 3 cc (60 mg), respectively. The intra- and inter- day precisions (RSD%) were less than 10.3%, while the accuracy was ranged from -7.34% to 9.10%. Extraction recovery ranged from 85.02 to 112.0% and the matrix effects ranged from 85.12% to 109.6%. The present method exhibited excellent linearity and the lower limits of quantification (LLOQ) were 30.0 ng/mL, 15.0 ng/mL, 80.0 ng/mL, 30.0 ng/mL, 10.0 ng/mL, 3.0 ng/mL, 2.5 ng/mL, 2.5 ng/mL, 1.5 ng/mL, 15.0 ng/mL, 75.0 ng/mL, 15.0 ng/mL, 30.0 ng/mL, and 20.0 ng/mL for gallic acid, protocatechuic acid, geniposidic acid, caffeic acid, ferulic acid, scopoletin, apigenin-7-o-glucuronide, daidzein, apigenin, ursolic acid, oleanolic acid, ß-sitosterol, coniferin, and stigmasterol, respectively. This analytical method was verified by the FDA guidelines for bioanalytical method validation and applied to investigate the pharmacokinetics and biodistribution of fourteen constituents of Hedyotis diffusa Willd extract in rats. These results provide useful information for improving the pharmacokinetics and biodistribution of fourteen bioactive ingredients of Hedyotis diffusa Willd extract in SD rats, supporting additional clinical application and Chinese herbal medicine safety evaluations.

The Antitumor Constituents from Hedyotis Diffusa Willd.

As a TCM, Hedyotis diffusa Willd. has been using to treat malignant tumors, and many studies also showed that the extracts from Hedyotis diffusa Willd. possessed evident antitumor activities. Therefore, we carried out chemical study on Hedyotis diffusa Willd. and investigated the cytotoxicity of the obtained compounds on a panel of eight tumor cell lines. As a result, four new compounds were isolated from Hedyotis diffusa Willd., including three iridoid glycosides of Shecaoiridoidside A-C (1-3) and a cerebroside of shecaocerenoside A (4). Also, six known iridoid compounds (5-10) were also obtained. The cytotoxicity of all compounds against human tumor cell lines of HL-60, HeLa, HCT15, A459, HepG2, PC-3, CNE-2, and BCG-823 were also evaluated in vitro. New compound 3 exhibited evident cytotoxicity to all tumor cell lines except the Hela, and the IC50 values are from 9.6 µM to 62.2 µM, while new compound 4 showed moderate cytotoxicity to all the cell lines, and the IC50 values are from 33.6 µM to 89.3 µM. By contrast, new compound 1 and known compound 9 showed moderate cytotoxicity to HCT15, A459, and HepG2 selectively. Known compound 7 also exhibited moderate cytotoxicity to HCT15 and A459 selectively.

Chloroform extract of Hedyotis diffusa Willd inhibits viability of human colorectal cancer cells via suppression of AKT and ERK signaling pathways.

Hedyotis diffusa Willd (HDW) is a widely used traditional Chinese medicine in clinical therapy to treat various types of cancer, including colorectal cancer (CRC), but its effective polar fractions and functional mechanisms remain unclear. The aim of the present study was to determine the most effective extract of HDW and to investigate its effects on the regulation of CRC cell proliferation and apoptosis, as well as to investigate the underlying molecular mechanisms. The results demonstrated that the chloroform extract of HDW (CEHDW) exhibited the most anticancer ability. Furthermore, results of the MTT assay, colony formation, carboxyfluorescein diacetate succinimidyl ester assay and annexin V/propidium iodide staining suggested that CEHDW significantly inhibits proliferation and promotes apoptosis in the SW620 CRC cell line. Additionally, reverse transcription-polymerase chain reaction and western blot analysis demonstrated that CEHDW treatment downregulated the expression of Survivin, proliferating cell nuclear antigen, Cyclin D1, cyclin-dependent kinase 4 and B-cell lymphoma 2 (Bcl-2), and upregulated the expression of Bcl-2-associated X protein at the mRNA and protein levels. CEHDW also decreased the phosphorylation of protein kinase B (AKT) and extracellular-signal-regulated kinase (ERK), which indicated that the suppression of the AKT and ERK signaling pathways may be one of the underlying molecular mechanisms by which CEHDW exhibited its anticancer effect. Thus, CEHDW may be a promising agent for anticancer therapy.

Hedyotis diffusa willd extract suppresses colorectal cancer growth through multiple cellular pathways.

The development of colorectal cancer (CRC) is strongly associated with the imbalance of various intracellular signal transduction cascades, including protein kinase B (AKT), mitogen-activated protein kinase 1 (MAPK), signal transducer and activator of transcription 3 (STAT3), as well as crosstalk between these signaling networks. At present, anti-tumor agents are often single-targeted and therefore are not always therapeutically effective. Moreover, long-term use of these anti-tumor agents often generates drug resistance and potential side effects. These problems highlight the urgent need for the development of novel and more effective anti-cancer drugs. Hedyotis diffusa Willd (HDW) has been used as a major component in traditional Chinese medicine for the clinical treatment of colorectal cancer, with a limited number of adverse effects. However, the molecular mechanisms, which underlie its anti-cancer activity, still require further elucidation. In the present study, using xenograft models and various different human CRC cell lines, the efficacy of the ethanol extract of HDW (EEHDW) against tumor growth was evaluated, and its underlying molecular mechanisms of action were investigated. It was demonstrated that EEHDW was able to inhibit cancer growth in vivo and in vitro. Furthermore, EEHDW was able to suppress the activation of several CRC-associated signaling pathways and was able to regulate the expression of various inflammatory and angiogenic factors. This resulted in the induction of apoptosis and inhibition of cellular proliferation, as well as tumor angiogenesis. The present study demonstrated that EEHDW is able to exhibit anti-cancer activity due to its ability to affect multiple intracellular targets, which suggests that it may be a novel multi-potent therapeutic agent for the treatment of colorectal cancer.