Synergistic action of Hedyotis diffusa Willd and Andrographis paniculata in Nasopharyngeal Carcinoma: Downregulating AKT1 and upregulating VEGFA to curb tumorigenesis.

OBJECTIVE: Nasopharyngeal carcinoma (NPC) remains a challenging cancer to treat. This study investigates the molecular mechanisms of Hedyotis diffusa Willd (HDW) combined with Andrographis paniculata (AP) in treating NPC. METHODS: Key compounds and target genes in HDW and AP were analyzed using network pharmacology. Protein-protein interaction (PPI) networks were constructed with STRING and visualized using Cytoscape. MCODE identified critical clusters, while DAVID facilitated GO and KEGG analyses. In vivo and in vitro experiments evaluated HDW-AP effects on NPC, including tumor volume, weight, Ki-67 expression, cell apoptosis, migration, invasion, cell cycle distribution, and DNA damage. RESULTS: The database identified 495 NPC-related genes and 26 compounds in the HDW-AP pair, targeting 165 genes. Fifty-eight potential therapeutic genes were found, leading to 18 key targets. KEGG analysis revealed a significant impact on 78 pathways, especially cancer pathways. Both in vivo and in vitro tests showed HDW-AP inhibited NPC cell proliferation, migration, invasion, and induced apoptosis. Mechanistically, this was achieved through AKT1 downregulation and VEGFA upregulation. CONCLUSION: The combination of HDW and AP targets 16 key genes to impede the development of NPC, primarily by modulating AKT1 and VEGFA pathways.

Identification of Hedyotis diffusa Willd-specific mRNA-miRNA-lncRNA network in rheumatoid arthritis based on network pharmacology, bioinformatics analysis, and experimental verification.

Hedyotis diffusa Willd (HDW) possesses heat-clearing, detoxification, anti-cancer, and anti-inflammatory properties. However, its effects on rheumatoid arthritis (RA) remain under-researched. In this study, we identified potential targets of HDW and collected differentially expressed genes of RA from the GEO dataset GSE77298, leading to the construction of a drug-component-target-disease regulatory network. The intersecting genes underwent GO and KEGG analysis. A PPI protein interaction network was established in the STRING database. Through LASSO, RF, and SVM-RFE algorithms, we identified the core gene MMP9. Subsequent analyses, including ROC, GSEA enrichment, and immune cell infiltration, correlated core genes with RA. mRNA-miRNA-lncRNA regulatory networks were predicted using databases like TargetScan, miRTarBase, miRWalk, starBase, lncBase, and the GEO dataset GSE122616. Experimental verification in RA-FLS cells confirmed HDW's regulatory impact on core genes and their ceRNA expression. We obtained 11 main active ingredients of HDW and 180 corresponding targets, 2150 RA-related genes, and 36 drug-disease intersection targets. The PPI network diagram and three machine learning methods screened to obtain MMP9, and further analysis showed that MMP9 had high diagnostic significance and was significantly correlated with the main infiltrated immune cells, and the molecular docking verification also showed that MMP9 and the main active components of HDW were well combined. Next, we predicted 6 miRNAs and 314 lncRNAs acting on MMP9, and two ceRNA regulatory axes were obtained according to the screening. Cellular assays indicated HDW inhibits RA-FLS cell proliferation and MMP9 protein expression dose-dependently, suggesting HDW might influence RA's progression by regulating the MMP9/miR-204-5p/MIAT axis. This innovative analytical thinking provides guidance and reference for the future research on the ceRNA mechanism of traditional Chinese medicine in the treatment of RA.

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.

Six new iridoid glycosides from the whole plants of Hedyotis diffusa.

Six new iridoid glycosides were isolated from the ethyl acetate fraction of the whole plants of Hedyotis diffusa Willd. They were identified as E-6-O-p-methoxycinnamoyl-10-O-acetyl scandoside acid methyl ester (1), Z-6-O-p-methoxycinnamoyl-10-O-acetyl scandoside acid methyl ester (2), E-6-O-caffeoyl scandoside methyl ester (3), E-6-O-p-coumaroyl-6'-O-acetyl scandoside methyl ester (4), Z-6-O-p-coumaroyl-6'-O-acetyl scandoside methyl ester (5), and E-6-O-p-coumaroyl-4'-O-acetyl scandoside methyl ester (6). The structures of them were elucidated based on unambiguous spectroscopic data (UV, IR, HRESIMS, and NMR). They were screened for anti-inflammatory effect, antioxidant effect, antitumor effect, and neuroprotective effect and did not show potent activities.

Network-based pharmacology-based research on the effect and mechanism of the Hedyotis diffusa-Scutellaria Barbata pair in the treatment of hepatocellular carcinoma.

The Hedyotis diffusa-Scutellaria officinalis pair (HD-SB) has therapeutic effects on a variety of cancers. Our study was to explore the mechanism of HD-SB in the treatment of hepatocellular carcinoma (HCC). A total of 217 active ingredients of HD-SB and 1196 HCC-related targets were reserved from the TCMSP and the SwissTarget Prediction database, and we got 63 intersection targets from GeneCards. We used a Venn diagram, and Cytoscape found that the three core ingredients were quercetin, luteolin, and baicalein. The PPI analysis showed that the core targets were TP53, CDK2, XPO1, and APP. Molecular docking results showed that these core ingredients had good binding potential with the core targets. HD-SB acts simultaneously on various HCC-related signaling pathways, including proteoglycans in cancer and the P53 signaling pathway. In vitro experiments confirmed that HD-SB can inhibit HepG2 cell proliferation by increasing TP53 and APP levels and decreasing XPO1 and CDK2 levels. This study analyzed active ingredients, core targets, and central mechanisms of HD-SB in the treatment of HCC. It reveals the role of HD-SB in targeting the P53 signaling pathway in the treatment of HCC. We hope that our research could provide a new perspective to the therapy of HCC and find new anticancer drugs.

Hedyotis diffusa-Sculellaria barbata (HD-SB) suppresses the progression of colorectal cancer cells via the hsa_circ_0039933/hsa-miR-204-5p/wnt11 axis.

Our previous study confirmed that the combination of Hedyotis diffusa (HD) and Scutellaria barbata (SB) significantly inhibited colorectal cancer cell proliferation and the WNT signaling pathway. However, the exact molecular modulation remains unclear. In this study, colorectal cancer cells (SW620) were treated with 1 mg/mL HD-SB for 24 h, and high-throughput sequencing of circRNAs was performed. The level of hsa_circ_0039933 in three colorectal cancer cell lines (HT-29, SW620, and HCT116) was verified by qPCR. After transfection of hsa_circ_0039933 overexpression plasmids or small interfering RNAs, CCK8, apoptosis, cell migration, and cell invasion were utilized to evaluate the function of hsa_circ_0039933 in the progression of colorectal cancer cells. We identified hsa_circ_0039933, which was downregulated in HD-SB-induced colorectal cancer cells and positively related to colorectal cancer progression. In SW620 cells with relatively high expression of hsa_circ_0039933, interfering with the expression of hsa_circ_0039933 inhibited the proliferation, invasion, and migration of SW620 cells. In HCT116 cells with relatively low expression of hsa_circ_0039933, overexpression of hsa_circ_0039933 promoted the proliferation and invasion and migration ability of HCT116. Mechanistically, hsa_circ_0039933 targeted hsa-miR-204-5p to increase the expression of wnt11, leading to the activation of the Wnt pathway, thereby promoting the proliferation of colorectal cancer cells. This work revealed the potential molecular mechanism of HD-SB for the treatment of colorectal cancer, which was to inhibit the Wnt signaling pathway through the hsa_circ_0039933/hsa-miR-204-5p/wnt11 axis, then suppressing proliferation, migration, and invasion in the colorectal cancer cell.

Exploring the mechanism of action of Hedyotis diffusa Willd on acne using network analysis.

In this study, we used a network pharmacological method to explore the active ingredients of Hedyotis diffusa Willd (HDW) in the treatment of acne and elucidated the physiological mechanisms in the human body in which they are involved. We identified the active compounds of HDW that are expected to act effectively in the human body using the Traditional Chinese Medicine Systems Pharmacology database and analysis platform and extracted potential interacting proteins for each active compound using the Swiss Target Prediction platform. Next, we analyzed the potential mechanisms of action of the protein targets shared by HDW and each standard drug on acne and assessed the possibility of spontaneous occurrence of the binding between proteins and active compounds through the molecular docking process. Seven active compounds were selected according to the oral bioavailability and drug-likeness criteria of the Traditional Chinese Medicine Systems Pharmacology database and analysis platform. Subsequently, 300 protein targets were collected from the Swiss Target Prediction. Using the Search Tool for the Retrieval of Interacting Genes/Proteins database, a protein-protein interaction network was constructed by analyzing the relationship between HDW, acne, and each standard drug. By analyzing the gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathway, the "positive regulation of lipid metabolic process" was found to be the most involved pathway shared by HDW, acne, and isotretinoin. An analysis of the protein targets shared by the antibiotic agents with HDW and acne found that "cholesterol storage" in tetracycline, "icosacoid transport" in azithromycin, "steroid hydroxylase activity" in erythromycin, "positive regulation of leukocyte tethering or rolling" in clindamycin, "response to UV-A" in minocycline, "steroid 11-beta-monooxygenase activity" in doxycycline, and "neutrophil-mediated immunity" in trimethoprim were the most involved. Virtual molecular docking analysis showed that all proteins spontaneously bound to their corresponding active compounds. Our analysis suggests that HDW can, directly and indirectly, suppress sebum secretion and exert antiinflammatory effects on acne. Further, HDW may regulate free radicals and suppress apoptosis. Therefore, HDW can be used as an alternative or supplement to standard drugs for acne treatment in patients who cannot use standard treatments due to side effects.

Network pharmacology and experimental validation to identify the potential mechanism of Hedyotis diffusa Willd against rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic, systemic, autoimmune disease that may lead to joint damage, deformity, and disability, if not treated effectively. Hedyotis diffusa Willd (HDW) and its main components have been widely used to treat a variety of tumors and inflammatory diseases. The present study utilized a network pharmacology approach, microarray data analysis and molecular docking to predict the key active ingredients and mechanisms of HDW against RA. Eleven active ingredients in HDW and 180 potential anti-RA targets were identified. The ingredients-targets-RA network showed that stigmasterol, beta-sitosterol, quercetin, kaempferol, and 2-methoxy-3-methyl-9,10-anthraquinone were key components for RA treatment. KEGG pathway results revealed that the 180 potential targets were inflammatory-related pathways with predominant enrichment of the AGE-RAGE, TNF, IL17, and PI3K-Akt signaling pathways. Screened through the PPI network and with Cytoscape software, RELA, TNF, IL6, TP53, MAPK1, AKT1, IL10, and ESR1 were identified as the hub targets in the HDW for RA treatment. Molecular docking was used to identify the binding of 5 key components and the 8 related-RA hub targets. Moreover, the results of network pharmacology were verified by vitro experiments. HDW inhibits cell proliferation in MH7A cells in a dose and time-dependent manner. RT-qPCR and WB results suggest that HDW may affect hub targets through PI3K/AKT signaling pathway, thereby exerting anti-RA effect. This study provides evidence for a clinical effect of HDW on RA and a research basis for further investigation into the active ingredients and mechanisms of HDW against RA.

Hedyocoronins A and B: two new oleanane saponins from the aerial parts of Hedyotis coronaria.

Two new oleanane saponins, hedyocoronin A (1) and hedyocoronin B (2), were isolated from the aerial parts of Hedyotis coronaria (Kurz) Craib, Rubiaceae, collected at Da Oai district, Lam Dong province in Vietnam. Their chemical structures were elucidated by HR-MS, 1D and 2D-NMR spectra, along with the comparison with those reported in the literature. Compounds 1 and 2 showed weak cytotoxicity against KB and HeLa-S3 cancer cell lines with IC50 values of more than 54 µM.

Hedyotis diffusa alleviate aflatoxin B1-induced liver injury in ducks by mediating Nrf2 signaling pathway.

Aflatoxin B1 (AFB1), the most harmful aflatoxins, is a frequent contamination in feed and food items, raising global concerns in animal production and human public health. Also, AFB1 induces oxidative stress, cytotoxicity, mutations, and DNA lesions through its metabolic transformation into aflatoxin B1-8,9-epoxide (AFBO) by cytochrome P450 (CYP450). Hedyotis diffusa (HD) is a traditional Chinese herbal medicine known for its multiple pharmacological activities, including antioxidant, anti-inflammatory, and immunomodulatory. Yet, the influence of HD on AFB1-induced liver injury in ducks is still unknown. Here, we investigated whether HD positively affects AFB1-induced liver injury in ducks. Results revealed that I) AFB1 caused significant changes in serum biochemical indices and decreased growth performance of ducks (such as ALT, AST, ALP, TP, ALB, final body weight, and body weight gain), whereas HD supplementation at 200 mg/kg mitigated these alterations. II) HD alleviated hepatic histopathological changes and liver index induced by AFB1 in ducks. III) HD significantly attenuated AFB1-induced oxidative stress, as measured by increased antioxidant enzyme activities such as SOD, GPx, and T-AOC and decreased MDA levels. Furthermore, HD reduced the level of AFB1-DNA adduct in duck liver. IV) HD significantly promoted the transcriptional expression of NF-E2-related nuclear factor 2 (Nrf2) and associated genes, including heme oxygenase 1 (HO-1), NAD(P)H dehydrogenase quinone 1 (NQO1), glutamate-cysteine ligase catalytic (GCLC). In conclusion, these results demonstrated that HD could activate the Nrf2 pathway in ducks to reduce the hepatotoxicity driven by AFB1. This finding also provides theoretical and data support for a deeper understanding of the toxic mechanisms of AFB1 and its prevention.

An anticomplement homogeneous polysaccharide from Hedyotis diffusa attenuates lipopolysaccharide-induced acute lung injury and inhibits neutrophil extracellular trap formation.

BACKGROUND: Owing to the involvement of the overactivated complement system in acute lung injury (ALI) development, anticomplement components may attenuate ALI. Hedyotis diffusa is a traditional Chinese medicine for treating lung heat and its crude polysaccharides (HDP) exhibit significant anticomplement activity in vitro. PURPOSE: To obtain an anticomplement homogeneous polysaccharide from HDP and verify its therapeutic effect and mechanism on ALI. METHODS: Diethylaminoethyl-52 (DEAE-52) cellulose and gel permeation columns were used to isolate a homogeneous polysaccharide HD-PS-3, which was then characterized using nuclear magnetic resonance (NMR) and methylation analysis. In vitro, the anticomplement activities of HD-PS-3 through classical and alternative pathways were determined using a hemolytic test. The therapeutic effects of HDP and HD-PS-3 on ALI were evaluated in lipopolysaccharide (LPS) intratracheal instilled mice. Hematoxylin and eosin (H&E) staining, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical staining were used to assess histological changes, measure cytokine levels, and evaluate the degree of complement component 3c (C3c) deposition and neutrophil infiltration, respectively. ELISA, western blotting, and immunofluorescence were used to analyze neutrophil extracellular trap (NET) formation. RESULTS: From HDP, 1.5 g of the homogeneous polysaccharide HD-PS-3 was obtained. HD-PS-3 was an acidic heteropolysaccharide with an acetyl group, which was composed of →4,6)-α-Glcp-(1→, →3,4)-α-Glcp-(1→, →4)-α-Glcp-(1→, →4,6)-α-Galp-(1→, →5)-α-Araf-(1→, α-Rhap-(1→, α-Araf-(1→, α-GlcpA-(1→, →4)-ß-Manp-(1→, ß-Manp-(1→ and →3)-ß-Manp-(1→. The in vitro results suggest that HD-PS-3 exhibited anticomplement activity with CH50 and AP50 values of 115 ± 12 µg/ml and 307 ± 11 µg/ml, respectively. After confirming the efficacy of HDP (200 mg/kg) in attenuating lung injury, the effect of HD-PS-3 on ALI was also investigated. HD-PS-3 (75 and 150 mg/kg) attenuated LPS-induced ALI as well, evidenced by lung pathology, lung injury scores, protein concentration, leukocyte counts, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) contents in bronchoalveolar lavage fluid (BALF). Mechanistically, HD-PS-3 inhibited complement activation, manifested in reduced pulmonary C3c deposition in lung tissue and complement component 3a (C3a) content in BALF. Neutrophil recruitment was also reduced by HD-PS-3, with significantly reduced pulmonary neutrophil infiltration and lower levels of C-X-C motif chemokine ligand 1 (CXCL1) and myeloperoxidase (MPO) in BALF. In addition, HD-PS-3 reduced the levels of MPO-DNA complex in BALF, decreased citrullinated histone H3 (Cit H3) expression and NET formation (colocalization of MPO, Cit H3, and DNA) in lung tissue. CONCLUSION: An anticomplement homogeneous polysaccharide HD-PS-3 was isolated from H. diffusa. HD-PS-3 exhibited a therapeutic effect against ALI, and the mechanism might be related to its inhibitory effects on complement activation, neutrophil recruitment, and NET formation.

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.

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.

Hedyotis diffusa injection induces ferroptosis via the Bax/Bcl2/VDAC2/3 axis in lung adenocarcinoma.

BACKGROUND: Lung cancer has the highest mortality rate among all cancer types. In combination with multiple chemotherapeutic options, traditional Chinese medicine has proven indispensable for the comprehensive treatment of lung cancer. PURPOSE: To investigate the effects of Hedyotis diffusa on lung adenocarcinoma cell lines and a BALB/c nude mouse xenograft model, and determine whether HDI could induce ferroptosis in lung adenocarcinoma cells along with the underlying mechanism. METHODS: The anti-tumor activity of HDI was determined in vitro by cell counting kit-8, clonogenic, and transwell assays. Subsequently, electron microscopy, a lipid reactive oxygen species assay, ferrous ion staining, and a malondialdehyde assay were performed to determine the effect on ferroptosis in lung adenocarcinoma cells. The mechanism was then further investigated using small molecule inhibitors, siRNA, and plasmid overexpression in vitro. Finally, the effects of HDI were assessed in tumor-bearing BALB/c nude mice, and HE staining was performed to observe tissue damage after HDI treatment. RESULTS: In vitro experiments showed that HDI could inhibit the viability of lung adenocarcinoma cells and induce lung adenocarcinoma cells ferroptosis via mechanisms independent of GPX4 and PUFA-PLS pathways but closely associated with VDAC2/3. HDI regulated VDAC2/3 activity by promoting Bax via inhibiting Bcl2, thereby inducing ferroptosis in lung adenocarcinoma cells. Furthermore, in vivo experiments showed that HDI significantly inhibited the growth of subcutaneous tumors in BALB/c nude mice with less organ damage and toxicity, and significantly increased the expression of the ferroptosis-related indicators 4HNE, TFR, and HMOX1 in tumor tissue. CONCLUSION: HDI can significantly reduce the survival of lung adenocarcinoma cells in vitro, inhibit the growth of subcutaneously transplanted tumors in BALB/c nude mice in vivo, and induce ferroptosis in lung adenocarcinoma cells via Bcl2 inhibition to promote Bax regulation of VDAC2/3.

Rapid fingerprint analysis based on supercritical fluid chromatography for quality evaluation of Hedyotis diffusa.

In this study, quality evaluations of Hedyotis diffusa (H. diffusa) batches by rapid fingerprint analysis based on supercritical fluid chromatography (SFC) were accomplished. Abundant chemical components of H. diffusa were effectively extracted by optimal supercritical fluid extraction conditions (20 % MeOH as modifier, 45 °C, 300 bar and 60 min). Then, the extract was separated by SFC on a Torus 1-AA column (100 × 3.0 mm i.d., 1.7 µm) within 10 min by gradient elution increasing from 5 % to 45 % modifier (MeOH containing 0.05 % TFA) at 1.2 mL/min, 30 °C and 2000 psi. The SFC approach exhibited short analysis time, while maintaining good peak shape and resolution. Seven major compounds were further identified by SFC coupled with tandem mass spectrometer to be phenylpropanoid, iridoids and anthraquinones. Finally, fingerprint analysis of 10 batches of H. diffusa by the developed SFC method was accomplished. The similarity values were between 0.894 and 0.968, indicating quality differences of H. diffusa from depending on origin and harvest year exist. The result demonstrates the feasibility of the SFC in batch quality evaluation of H. diffusa.

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.

Hedyotis diffusae Herba-Andrographis Herba inhibits the cellular proliferation of nasopharyngeal carcinoma and triggers DNA damage through activation of p53 and p21.

Dysregulation of the cell cycle and the resulting aberrant cellular proliferation has been highlighted as a hallmark of cancer. Certain traditional Chinese medicines can inhibit cancer growth by inducing cell cycle arrest. In this study we explore the effect of Hedyotis diffusae Herba-Andrographis Herba on the cell cycle of nasopharyngeal carcinoma (NPC). Hedyotis diffusae Herba-Andrographis Herba-containing serum was prepared and then added to the cell culture medium. BrdU, comet, and FUCCI assays, western blot analysis and flow cytometry analysis revealed that Hedyotis diffusae Herba-Andrographis Herba treatment significantly alters cell proliferation, DNA damage, and cell cycle distribution. Xenograft mouse model experiments were performed, confirming these in vitro findings in vivo. Treatment with Hedyotis diffusae Herba-Andrographis Herba inhibited cell proliferation, promoted DNA damage, and arrested NPC cells progression from G1 to S phase. Further examination of the underlying molecular mechanisms revealed that treatment with Hedyotis diffusae Herba-Andrographis Herba increased the expression of p53 and p21, while reducing that of CCND1, Phospho-Rb, E2F1, γH2AX, and Ki-67 both in vivo and in vitro. Conversely, the inhibition of p53 and p21 could abolish the promoting effect of Hedyotis diffusae Herba-Andrographis Herba on the NPC cell cycle arrest at the G1 phase, contributing to the proliferation of NPC cells. Hedyotis diffusae Herba-Andrographis Herba suppressed the tumor growth in vivo. Overall, these findings suggest that Hedyotis Diffusae Herba-Andrographis prevent the progression of NPC by inducing NPC cell cycle arrest at the G1 phase through a p53/p21-dependent mechanism, providing a novel potential therapeutic treatment against NPC.

Dose-dependent effects of Hedyotis diffusa extract on the pharmacokinetics of tamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen.

Tamoxifen, a widely prescribed medication in premenopausal women diagnosed with hormone-dependent breast cancer, is potentially co-prescribed with Hedyotis diffusa (H. diffusa), particularly in Taiwan. However, no related report has investigated the drug-herb interaction of H. diffusa on the pharmacokinetics of tamoxifen and its metabolites. In the present study, male Sprague-Dawley rats were administered different doses of H. diffusa extract for 5 consecutive days prior to the administration of tamoxifen (10 mg/kg). A validated ultra-liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system was developed to monitor tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen, and endoxifen in rat plasma. Pharmacokinetic results demonstrated that the area under curves (AUCs) of tamoxifen and the relative bioavailability (%) of tamoxifen were dose-dependently decreased (31-68%) by pre-treatment with H. diffusa extract (3 g/kg and 6 g/kg). In addition, the conversion ratio of 4-hydroxytamoxifen was downregulated (0.5-fold change) and the N-desmethyltamoxifen conversion ratio was upregulated (2-fold change) by high-dose H. diffusa extract. As a result, the relative bioavailability and biotransformation changes affect the clinical efficacy of tamoxifen treatment. These preclinical findings reveal a hitherto unreported interaction between tamoxifen and H. diffusa extract that has implications for their therapeutic efficacy in treating breast cancer.

[Effect of "Hedyotis Diffusae Herba-Smilacis Glabrae Rhizoma" in treatment of lung adenocarcinoma based on network pharmacology].

To explore the mechanism of Hedyotis Diffusae Herba-Smilacis Glabrae Rhizoma(HDH-SGR) in treating lung adenocarcinoma based on big data bioinformatics combined with network pharmacology analysis and molecular docking technology. The chemical components and potential therapeutic targets of HDH-SGR were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). Lung adenocarcinoma-related genes were obtained from The Cancer Genome Atlas(TCGA), Therapeutic Target Database(TTD), Pharmacogenetics and Pharmacogenomics Knowledge Base(PharmGKB), Online Mendelian Inheritance in Man(OMIM), DrugBank, and GeneCards. "Drug component-target" network was constructed using Cytoscape to screen out key compounds. STRING was used to build protein-protein interaction(PPI) network and core targets were screened out by Cytoscape-CytoNCA topology analysis. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) analyses of target genes were performed by R-clusterProfiler. Finally, key compounds were docked to core target genes using AutoDock. The results showed that 22 active compounds and 499 potential therapeutic targets were obtained from HDH-SGR. A total of 14 332 lung adenocarcinoma-related targets were screened out through six data platforms, including 182 common targets. Fifteen core targets were screened out from the PPI network. GO and KEGG analyses revealed significant enrichment of relevant target genes in various biological processes, cellular functions(e.g., response to lipopolysaccharide, nuclear receptor activity, and ligand-activated transcription factor activity) and close relationship between target genes and non-small cell lung cancer signaling pathways. Based on the results of molecular docking validation, diosgenin, quercetin, naringenin, taxifolin, 2-methoxy-3-methyl-9,10-anthraquinone, stigmasterol, and ß-sitosterol were able to bind tightly to the core targets. HDH-SGR can intervene in lung adenocarcinoma through multiple targets and signaling pathways, such as non-small cell lung cancer signaling pathways. The binding of active components in Chinese medicine to key targets is presumedly one of the mechanisms that produce therapeutic effects.

[Effects of Hedyotis diffusa polysaccharide extract on autophagy of endoplasmic reticulum in laryngeal cancer Hep-2 cells].

Objective: To investigate the effects of Hedyotis diffusa polysaccharide extract (HDPE) on endoplasmic reticulum autophagy in laryngeal cancer Hep-2 cells. Methods: The cells were divided into control group, HDPE 100, 200, 400 mg/L group and 3-MA(autophagy inhibitor) group. MTT assay was used to detect the inhibition rate of cell proliferation after 24, 48 and 72 h of culture, and TUNEL was used to detect the apoptosis of cells in each group. The changes of autophages and autophagic lysosomes were observed by MDC staining, and the formation of autophagic vesicles around endoplasmic reticulum was observed by transmission electron microscopy. Western blot was used to detect the expressions of Beclin-1, microtubule-associated light chain protein 3I (LC3I), microtubule-associated light chain protein 3II (LC3II), glucose regulatory protein 78 (GRP78), activated transcription factor 6 (ATF6) and CCAAT enhancer binding protein homologous protein (CHOP) in each group. Results: Compared with the control group, the inhibition rate of cell proliferation and apoptotic index AI in HDPE 100, 200, 400 mg/L group and blocker group were increased, the positive rate of MDC was decreased, and the autophagic vesicles around endoplasmic reticulum were reduced. The expression levels of GRP78, ATF6 and CHOP and the ratio of LC3I/LC3II were increased, and the expression of Beclin-1 was decreased (P＜0.05). Compared with 3-MA group, the inhibition rate of cell proliferation and apoptotic index AI in HDPE 400 mg/L group were increased, the positive rate of MDC was decreased, the expressions of GRP78, ATF6 and CHOP and LC3I/LC3II ratio were increased, and the expression of Beclin-1 was decreased (P＜0.05). Conclusion: HDPE may inhibit the proliferation of Hep-2 cells by inhibiting endoplasmic reticulum autophagy and promoting the apoptosis of endoplasmic reticulum stress in laryngeal cancer Hep-2 cells.