The immunotherapy mechanism of Hedyotis Diffusae Herba in treating liver cancer: a study based on network pharmacology, bioinformatics, and experimental validation.

Liver cancer is a malignant tumor that develops on or inside the liver. Hedyotis diffusa Willd (HDW) plays a significant role in anti-tumor activities; however, its mechanism against liver cancer remains unclear. This study aims to evaluate the immunotherapeutic mechanism of HDW in treating liver cancer through network pharmacology, bioinformatics analysis, and experimental validation. Network pharmacology was utilized to identify the active components and potential targets of HDW from the TCMSP database. A potential target protein-protein interaction (PPI) network was constructed using the STRING database, followed by function and pathway enrichment analysis of the targets using GO and KEGG methods. In addition, the key targets for HDW against liver cancer were identified using five different algorithms in Cytoscape. The TCGA and HPA databases were used to assess the mRNA and protein expression of core target genes in normal liver and liver cancer tissues and their relationship with overall survival in liver cancer, as well as their role in immune infiltration. Molecular docking between the core components of HDW and the core targets was performed using PyMOL software. The effects of HDW on the proliferation and apoptosis of liver cancer cells were examined using MTT and flow cytometry. The regulatory effects of the core component quercetin on core targets were validated using RT-qPCR and Western blot. A total of 163 potential targets were identified by searching for intersections among 7 types of active components and all potential and liver cancer targets. PPI network analysis revealed the core targets IL6 and TNF. GO enrichment analysis involved 2089 biological processes, 76 cellular components, and 196 molecular functions. KEGG enrichment analysis suggested that the anti-cancer effects of HDW might be mediated by the AGE-RAGE signaling pathway, IL-17 signaling pathway, TNF signaling pathway, PI3K-Akt signaling pathway, and NF-κB signaling pathway. Database validation of key targets showed that mRNA and protein expression results for the IL6 gene were contradictory, while those for the TNF gene were consistent, both being underexpressed in liver cancer. Importantly, the expression of IL6 and TNF was related to the infiltration of 24 types of immune cells, with the highest correlation with macrophages. Molecular docking showed that IL6 and TNF had high binding stability with quercetin, with binding energies of - 7.4 and - 6.0 kJ∙mol-1, respectively. Experimental validation showed that quercetin inhibited liver cancer cell proliferation and promoted apoptosis in a dose-dependent manner, with protein results indicating that quercetin downregulated the mRNA and protein expression of IL6 and TNF, and upregulated key proteins in the AGE-RAGE signaling pathway, AGEs, and RAGE. This study comprehensively elucidates the activity, potential targets, and molecular mechanisms of HDW against liver cancer, providing a promising strategy for the scientific basis and treatment mechanism of traditional Chinese medicine in treating liver cancer.

Network pharmacology prediction and experiment validation of anti-liver cancer activity of Curcumae Rhizoma and Hedyotis diffusa Willd.

Objective: This study aims to elucidate anti-liver cancer components and potential mechanisms of Curcumae Rhizoma and Hedyotis diffusa Willd (CR-HDW). Methods: Effective components and targets of CR-HDW were identified from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Liver cancer-related genes were collected from GeneCards, Gene-Disease Association (DisGeNET), and National Center for Biotechnology Information (NCBI). Protein-protein interaction networks, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were conducted to analyze the identified genes. Molecular docking was used to simulate binding of the active components and their target proteins. Cell activity assay, western blot, and senescence-associated ß-galactosidase (SA-ß-gal) experiments were conducted to validate core targets identified from molecular docking. Results: Ten active compounds of CR-HDW were identified including quercetin, 3-epioleanic acid and hederagenin. The primary core proteins comprised Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Protein Kinase B(AKT1), etc. The pathways for Phosphoinositide 3-kinase (PI3K)/ AKT, cellular senescence, Fork head boxO (FOXO) were revealed as important for anti-cancer activity of CR-HDW. Molecular docking demonstrated strong binding between liver cancer target proteins and major active components of CR-HDW. In-vitro experiments confirmed that hederagenin and 3-epioleolic acid inhibited HuH-7 cell growth, reduced expression of PI3K, AKT, and mechanistic target of rapamycin (mTOR) proteins. Hederagenin also induced HuH-7 senescence. Conclusions: In summary, The authors' results suggest that the CR-HDW component (Hederagenin, 3-epoxy-olanolic acid) can inhibit the proliferation of HuH-7 cells by decreasing PI3K, AKT, and mTOR. Hederagenin also induced HuH-7 senescence.

Molecular mechanism of the treatment of lung adenocarcinoma by Hedyotis Diffusa: an integrative study with real-world clinical data and experimental validation.

Background: With a variety of active ingredients, Hedyotis Diffusa (H. diffusa) can treat a variety of tumors. The purpose of our study is based on real-world data and experimental level, to double demonstrate the efficacy and possible molecular mechanism of H. diffusa in the treatment of lung adenocarcinom (LUAD). Methods: Phenotype-genotype and herbal-target associations were extracted from the SymMap database. Disease-gene associations were extracted from the MalaCards database. A molecular network-based correlation analysis was further conducted on the collection of genes associated with TCM and the collection of genes associated with diseases and symptoms. Then, the network separation SAB metrics were applied to evaluate the network proximity relationship between TCM and symptoms. Finally, cell apoptosis experiment, Western blot, and Real-time PCR were used for biological experimental level validation analysis. Results: Included in the study were 85,437 electronic medical records (318 patients with LUAD). The proportion of prescriptions containing H. diffusa in the LUAD group was much higher than that in the non-LUAD group (p < 0.005). We counted the symptom relief of patients in the group and the group without the use of H. diffusa: except for symptoms such as fatigue, palpitations, and dizziness, the improvement rate of symptoms in the user group was higher than that in the non-use group. We selected the five most frequently occurring symptoms in the use group, namely, cough, expectoration, fatigue, chest tightness and wheezing. We combined the above five symptom genes into one group. The overlapping genes obtained were CTNNB1, STAT3, CASP8, and APC. The selection of CTNNB1 target for biological experiments showed that the proliferation rate of LUAD A549 cells in the drug intervention group was significantly lower than that in the control group, and it was concentration-dependent. H. diffusa can promote the apoptosis of A549 cells, and the apoptosis rate of the high-concentration drug group is significantly higher than that of the low-concentration drug group. The transcription and expression level of CTNNB1 gene in the drug intervention group were significantly decreased. Conclusion: H. diffusa inhibits the proliferation and promotes apoptosis of LUAD A549 cells, which may be related to the fact that H. diffusa can regulate the expression of CTNNB1.

Network pharmacology and experimental verification to explore the effect of Hedyotis diffusa on Alzheimer's disease.

This study aimed to explore the active components and the effect of Hedyotis diffusa (HD) against Alzheimer's disease (AD) via network pharmacology, molecular docking, and experimental evaluations. We conducted a comprehensive screening process using the TCMSP, Swiss Target Prediction, and PharmMapper databases to identify the active components and their related targets in HD. In addition, we collected potential therapeutic targets of AD from the Gene Cards, Drugbank, and OMIM databases. Afterward, we utilized Cytoscape to establish both protein-protein interaction (PPI) networks and compound-target (C-T) networks. To gain further insights into the functional aspect, we performed GO and KEGG pathway analyses using the David database. Next, we employed Autodock vina to estimate the binding force between the components and the hub genes. To validate our network pharmacology findings, we conducted relevant experiments on Caenorhabditis elegans, further confirming the reliability of our results. Then a total of six active compounds and 149 therapeutic targets were detected. Through the analysis of the association between active compounds, therapeutic targets, and signaling pathways, it was observed that the therapeutic effect of HD primarily encompassed the inhibition of Aß, suppression of AChE activity, and mitigating oxidative stress. Additionally, our investigation revealed that the key active compounds in HD primarily consisted of iridoids, which exhibited resistance against AD by acting on the Alzheimer's disease pathway and the AGE-RAGE signaling pathway in diabetic complications.

Hedyotis diffusa Willd inhibits inflammation and oxidative stress to protect against chronic prostatitis via the NRF2/ARE signaling pathway.

Chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) is a common and serious disease with unclear pathogenesis and recurrent symptoms. Hedyotis diffusa Willd (HDW) has been recognized for its potential in managing various chronic inflammatory diseases. This research aimed to interrogate the mechanism of HDW in treating CP/CPPS. Complete Freund Adjuvant (CFA) and LPS were utilized to establish the rat and cell models of CP/CPPS. Results showed that HDW decreased levels of inflammation-related factors in CP rat prostate tissue and LPS-elicited RWPE-1 cell injury model. Moreover, HDW administration impaired oxidative stress in the prostate and RWPE-1 cells. In addition, HDW treatment activated the NRF2/ARE signaling in rat prostate tissue and cell models. Interestingly, NRF2/ARE pathway inhibitor ML385 reversed the inhibition effects of cell apoptosis, inflammation, and oxidative stress triggered by HDW. In summary, HDW alleviated inflammation and oxidative stress by activating NRF2/ARE signaling in CP/CPPS rat model and human prostate epithelial cell injury model.

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.

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.

Network pharmacology and experimental verification study on the mechanism of Hedyotis diffusa Willd in treating colorectal cancer.

This study aimed to evaluate the pharmacological mechanism of Hedyotis diffusa Willd against CRC (colorectal cancer) using network pharmacological analysis combined with experimental validation. The active components and potential targets of Hedyotis diffusa Willd were screened from the tax compliance management program public database using network pharmacology. The core anti-CRC targets were screened using a protein-protein interaction (PPI) network. The mRNA and protein expression of core target genes in normal colon and CRC tissues and their relationship with overall CRC survival were evaluated using The Cancer Genome Atlas (TCGA), Human Protein Atlas (HPA), and Gene Expression Profiling Interactive Analysis (GEPIA) databases. Functional and pathway enrichment analyses of the potential targets were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The first six core targets with stable binding were molecular-docked with the active components quercetin and ß-sitosterol. Finally, the results of network pharmacology were verified using in vitro experiments. In total, 149 potential targets were identified by searching for seven types of active components and the intersection of all potential and CRC targets. PPI network analysis showed that ten target genes, including tumor protein p53 (TP53) and recombinant cyclin D1 (CCND1), were pivotal genes. GO enrichment analysis involved 2043 biological processes, 52 cellular components, and 191 molecular functions. KEGG enrichment analysis indicated that the anticancer effects of H. alba were mediated by tumor necrosis factor, interleukin-17, and nuclear factor-κB (NF-κB) signaling pathways. Validation of key targets showed that the validation results for most core genes were consistent with those in this study. Molecular docking revealed that the ten core target proteins could be well combined with quercetin and ß-sitosterol and the structure remained stable after binding. The results of the in vitro experiment showed that ß-sitosterol inhibited proliferation and induced apoptosis in SW620 cells. This study identified a potential target plant for CRC through network pharmacology and in vitro validation.

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.

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.

Seventeen undescribed iridoid derivatives with anti-inflammatory effects from Hedyotis diffusa and their structure-activity relationships.

Seventeen undescribed iridoid derivatives (1-17) and four known compounds (18-21) were isolated from the whole plant of Hedyotis diffusa Willd. Their structures were elucidated based on unambiguous spectroscopic data (UV, IR, HRESIMS, CD, and 1D and 2D NMR). It is noteworthy that compounds 1-8, which possess unique long-chain aliphatic acid moiety, were reported for the first time among the iridoid natural products. All compounds were evaluated for their anti-inflammatory activities in lipopolysaccharide-induced RAW 264.7 cells. Compounds 2, 4, and 6 showed significant suppression effects on nitric oxide production, with IC50 values of 5.69, 6.16, and 6.84 µM, respectively. The structure-activity relationships of these compounds indicated that long-chain aliphatic moieties at C-10 might be the key group for their anti-inflammatory activities. The therapeutic properties of these iridoid derivatives could give an insight into utilizing H. diffusa as a natural source of anti-inflammatory agents.

Ethyl Acetate Fraction from Hedyotis Diffusa Plus Scutellaria Barbata Inhibits the Progression of Breast Cancer via Targeting LMO1 and AKT/Mtor Signaling Pathway

BACKGROUND: Traditional Chinese medicines are widely used in cancer treatment. Scutellaria barbata and Hedyotis diffusa herb pair (SH) has an anticancer effects in various tumors. However, the specific mechanism of SH in breast cancer remains unclear. METHODS: In the present research, we investigated the effect and regulatory network of SH in in breast cancer. CCK8, colony formation, transwell, wound healing and flow cytometry analysis were used for the detection of cell function. RESULTS: Ethyl acetate fraction from SH at an equal weight ratio (EA11) could inhibit the proliferation, migration and invasion of MCF7 and MDA-MB-231 cells. It also induced apoptosis in these two cell lines by downregulating Bcl2 and upregulating Bax and Cleaved-Caspase3. SH reduced the activation of the AKT/mTOR signaling pathway and the expression of p70S6K. Sequencing results showed that LMO1 was significantly downregulated in SH-treated cells compared with control cells. Importantly, overexpression of LMO1 attenuated the inhibitory effect of SH on cell proliferation and invasion and induced inflammatory tumor microenvironment. CONCLUSION: In conclusion, the SH herb pair inhibited the proliferation and metastasis through downregulating LMO1 expression and reducing the activation of the AKT/mTOR signaling pathway. LMO1 has the potential as a new target in the treatment of breast cancer.

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.

Efficient strategies for preparative separation of iridoid glycosides and flavonoid glycosides from Hedyotis diffusa.

Efficient strategies for the preparative separation of iridoid glycosides and flavonoid glycosides from Hedyotis diffusa using preparative high-performance liquid chromatography combined with appropriate pretreatment technologies were developed. Four fractions (Fr.1-1, Fr.1-2, Fr.1-3, and Fr.2-1) were firstly isolated from the crude extract of Hedyotis diffusa by column chromatography with C18, resin, and silica gel materials, respectively. Then, corresponding separation strategies were developed according to the polarity and chemical constituents. High-polar compounds of Fr.1-1 were purified by hydrophilic reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography mode. The combination of C18 and phenyl columns realized the complementary separation of iridoid glycosides in Fr.1-2. Meanwhile, the improved selectivity caused by the change of organic solvent in the mobile phase was utilized to realize the purification of flavonoid glycosides in Fr.1-3 and Fr. 2-1. Finally, 27 compounds (purity > 95%) mainly involving nine iridoid glycosides and five flavonoid glycosides were obtained. A complete strategy was established for the separation of a complex sample with a wide polarity range, to jointly solve the problems of enrichment of target components and separation of structural analogs.

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.

Two new iridoid glycosides from Hedyotis diffusa.

Two new iridoid glycosides, named productasperulosidic acid butyl ester (1) and E-6-O-3-hydroxy-p-methoxycinnamoyl scandoside methyl ester (2), along with nine known ones (3-11), were isolated from Hedyotis diffusa Willd. The structures of them were elucidated by extensive 1D, 2D NMR and HR-ESI-MS spectral data. Compounds 1-11 showed no significant cytotoxic activity against HeLa cells.

Inhibitory effects of total triterpenoids isolated from the Hedyotis diffusa willd on H1975 cells.

It is estimated that non-small-cell lung cancer (NSCLC) is responsible for 80% of human deaths related to lung cancer worldwide. Currently, it has been discovered that two transcription factors. Nuclear factor-κB (NF-κB) and Signal transducer and activator of transcription 3 (STAT3) were the main factors affecting inflammation and cancer, and their activation promoted lung cancer cell proliferation. Hedyotis diffusa Willd. (H. diffusa) is an herbal Chinese medicine, which has always been used for the treatment of malignant tumors in clinical. Previous research found that H. diffusa could inhibit the proliferation of H1975 cells, but the specific mechanisms remain elusive. We investigated the effects of total triterpenes extracted from H. diffusa (TTH) on the migration, proliferation and apoptosis of H1975 cells. Cell-cycle and immunofluorescence analysis showed that TTH could block H1975 cells at G0/G1 phase and induce apoptosis of experimental cells. The protein levels of Bcl-2 were decreased, while the levels of pro-apoptotic Bax were increased. In addition, TTH could also inhibit the migration of H1975 cells through downregulated MMP-2 and MMP-9 and upregulated TIMP-2. Further research found that the level of phospho-STAT3 was significantly decreased after administration of TTH. And protein expression level of NF-κB in nucleus was decreased after TTH treatment, while NF-κB in cytoplasm increased. These results suggested that TTH could inhibit the proliferation and migration of H1975 cells, and also could induce cell apoptosis. These effects were closely connected to the activation of NF-κB and the phosphorylation of STAT3.

Novel Antiviral Efficacy of Hedyotis diffusa and Artemisia capillaris Extracts against Dengue Virus, Japanese Encephalitis Virus, and Zika Virus Infection and Immunoregulatory Cytokine Signatures.

Currently, there are no specific therapeutics for flavivirus infections, including dengue virus (DENV) and Zika virus (ZIKV). In this study, we evaluated extracts from the plants Hedyotis diffusa (HD) and Artemisia capillaris (AC) to determine the antiviral activity against DENV, ZIKV, and Japanese encephalitis virus (JEV). HD and AC demonstrated inhibitory activity against JEV, ZIKV, and DENV replication and reduced viral RNA levels in a dose-responsive manner, with non-cytotoxic concentration ranging from 0.1 to 10 mg/mL. HD and AC had low cytotoxicity to Vero cells, with CC50 values of 33.7 ± 1.6 and 30.3 ± 1.7 mg/mL (mean ± SD), respectively. The anti-flavivirus activity of HD and AC was also consistent in human cell lines, including human glioblastoma (T98G), human chronic myeloid leukemia (K562), and human embryonic kidney (HEK-293T) cells. Viral-infected, HD-treated cells demonstrated downregulation of cytokines including CCR1, CCL26, CCL15, CCL5, IL21, and IL17C. In contrast, CCR1, CCL26, and AIMP1 were elevated following AC treatment in viral-infected cells. Overall, HD and AC plant extracts demonstrated flavivirus replication inhibitory activity, and together with immunoregulatory cytokine signatures, these results suggest that HD and AC possess bioactive compounds that may further be refined as promising candidates for clinical applications.

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.