Neferine inhibits BMECs pyroptosis and maintains blood-brain barrier integrity in ischemic stroke by triggering a cascade reaction of PGC-1α.

Blood-brain barrier disruption is a critical pathological event in the progression of ischemic stroke (IS). Most studies regarding the therapeutic potential of neferine (Nef) on IS have focused on neuroprotective effect. However, whether Nef attenuates BBB disruption during IS is unclear. We here used mice underwent transient middle cerebral artery occlusion (tMCAO) in vivo and bEnd.3 cells exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) injury in vitro to simulate cerebral ischemia. We showed that Nef reduced neurobehavioral dysfunction and protected brain microvascular endothelial cells and BBB integrity. Molecular docking, short interfering (Si) RNA and plasmid transfection results showed us that PGC-1α was the most binding affinity of biological activity protein for Nef. And verification experiments were showed that Nef upregulated PGC-1α expression to reduce mitochondrial oxidative stress and promote TJ proteins expression, further improves the integrity of BBB in mice. Intriguingly, our study showed that neferine is a natural PGC-1α activator and illustrated the mechanism of specific binding site. Furthermore, we have demonstrated Nef reduced mitochondria oxidative damage and ameliorates endothelial inflammation by inhibiting pyroptosis to improve BBB permeability through triggering a cascade reaction of PGC-1α via regulation of PGC-1α/NLRP3/GSDMD signaling pathway to maintain the integrity of BBB in ischemia/reperfusion injury.

Angelica Yinzi Alleviates Pruritus-Related Atopic Dermatitis through Skin Repair, Antioxidation, and Balancing Peripheral µ- and κ-opioid Receptors.

Background: Angelica Yinzi (AYZ) is a Chinese traditional herbal formula reported to attenuate itches and inflammation caused by atopic dermatitis (AD). However, the underlying mechanism of AYZ in the attenuation of itchiness and inflammation remains unknown. Objective: This study investigated the mechanism of AYZ in reducing itchiness in mice with 1-chloro-2,4-dinitrobenzene- (DNCB-)-induced atopic dermatitis. Methods: Hematoxylin and eosin (H&E) and toluidine blue staining were used to evaluate pathological changes in skin tissue, while an enzyme-linked immunosorbent assay (ELISA) was used to assess the cytokine levels in the skin. After that, qRT-PCR was performed to determine the mRNA levels of cytokines in the skin. Immunofluorescence and western blotting analysis were further used to assess µ-opioid receptor (MOR) expression and immunohistochemistry to assess the p-ERK, p-AKT, and κ-opioid receptor (KOR). Results: The AYZ treatment alleviated the AD clinical symptoms, including decreasing the scratching frequency, the ear thickness, and the infiltration of mast cells, lymphocytes, inflammatory cells, and mononuclear cells. In addition, AYZ inhibited the expression of interleukin (IL)-13, thymic stromal lymphopoietin (TSLP), and reduced neuraminidase (NA), corticotropin-releasing factor (CRF), and reactive oxygen species (ROS) expression. Markers involved in itches, such as p-ERK and p-AKT, were significantly downregulated following AYZ treatment. Besides, AYZ significantly increased MOR expression and downregulated KOR in the epidermis and spinal cord. Conclusion: Our findings imply that AYZ ameliorates pruritus-related AD through skin repair, antioxidation, and balancing peripheral MOR and KOR. The findings in this study lay a theoretical foundation for the control mechanism of peripheral itch.

Tetrandrine inhibits the proliferation of mesangial cells induced by enzymatically deglycosylated human IgA1 via IgA receptor/MAPK/NF-κB signaling pathway.

Objective: Despite the use of renin-angiotensin system blockade and immunosuppressive drugs, including corticosteroids, the current treatment regimens for Immunoglobulins A nephropathy (IgAN) are severely limited. The proliferation of mesangial cell and deposition of deglycosylated human IgA1 immune complex are the most common pathologic features of IgAN. We examined the tetrandrine potential of suppressing the proliferation of mesangial cells and explored its underlying mechanisms with a focus on IgA receptor/MAPK/NF-κB signaling pathway. Methods: Standard human IgA (native IgA) were enzymatically desialylated (deS IgA) or further degalactosylated (deS/deGal IgA) using neuraminidase and ß-galactosidase. Rat glomerular mesangial cells (HBZY-1) and human renal mesangial cells (HRMC) stimulated by IgA were used to observe the suppressive effect of tetrandrine. The MTT assay was used to detect the cell viability. The protein expression of IgA receptor/MAPK/NF-κB signaling pathway was examined by Western blot. Cell cycle analysis was measured by flow cytometer. Results: Native IgA and deS IgA showed limited stimulation effect on both HBZY-1 cells and HRMCs, whereas deS/deGal IgA significantly stimulated the proliferation of both HBZY-1 cells and HRMCs (p < 0.05). Compared with non-stimulation of deS/deGal IgA, 1-3 µM of tetrandrine had stronger inhibitory effect on the proliferation of HBZY-1 cells and HRMCs with the stimulation of deS/deGal IgA (p < 0.05), suggesting that tetrandrine possibly inhibited the proliferation of mesangial cells induced by deglycosylated human IgA1 specifically. Molecular mechanism study revealed that tetrandrine decreased the expression of IgA1 receptor, CD71 and ß4GALT1, and inhibited the activation of MAPK/NF-κB significantly (p < 0.05). Moreover, these inhibitory effect of tetrandrine caused cell cycle arrest and stopped the cell growth in the S phase companied with the upregulating of cyclin A2 and downregulating of cyclin D1. Conclusion: Taken together, tetrandrine inhibited the proliferation of mesangial cells induced by enzymatically deglycosylated human IgA1 via IgA receptor/MAPK/NF-κB signaling pathway. Based on these potential molecular mechanisms, tetrandrine would be an appealing therapeutic option for IgAN.

Screening the effective components in treating dampness stagnancy due to spleen deficiency syndrome and elucidating the potential mechanism of Poria water extract.

Poria is an important medicine for inducing diuresis to drain dampness from the middle energizer. However, the specific effective components and the potential mechanism of Poria remain largely unknown. To identify the effective components and the mechanism of Poria water extract (PWE) to treat dampness stagnancy due to spleen deficiency syndrome (DSSD), a rat model of DSSD was established through weight-loaded forced swimming, intragastric ice-water stimulation, humid living environment, and alternate-day fasting for 21 days. After 14 days of treatment with PWE, the results indicated that PWE increased fecal moisture percentage, urine output, D-xylose level and weight; amylase, albumin, and total protein levels; and the swimming time of rats with DSSD to different extents. Eleven highly related components were screened out using the spectrum-effect relationship and LC-MS. Mechanistic studies revealed that PWE significantly increased the expression of serum motilin (MTL), gastrin (GAS), ADCY5/6, p-PKAα/ß/γ cat, and phosphorylated cAMP-response element binding protein in the stomach, and AQP3 expression in the colon. Moreover, it decreased the levels of serum ADH, the expression of AQP3 and AQP4 in the stomach, AQP1 and AQP3 in the duodenum, and AQP4 in the colon. PWE induced diuresis to drain dampness in rats with DSSD. Eleven main effective components were identified in PWE. They exerted therapeutic effect by regulating the AC-cAMP-AQP signaling pathway in the stomach, MTL and GAS levels in the serum, AQP1 and AQP3 expression in the duodenum, and AQP3 and AQP4 expression in the colon.

Screening of anti-liver fibrosis peptides from turtle shell protein using two-enzyme hydrolysis by molecular docking.

Turtle shell as a food residue of Pelodiscus sinensis (a type of edible aquatic animal) is widely used in Traditional Chinese Medicine for hepatic fibrosis therapy. Previous studies have demonstrated that the peptides (<6 kDa) derived from turtle shells are considered effective components. The protein of turtle shells has important potential as a source of bioactive peptides which may play a role as ingredients in functional foods. In the present study, the protein of turtle shell was hydrolyzed using a two-enzyme combination. It was found that the hydrolysates obtained by a combination of pepsin and trypsin showed the highest anti-liver fibrosis activity relative to other combinations in a cell viability assay. The hydrolysates were separated and purified by ultra-filtration (<6 kDa), gel filtration chromatography (GFC) and high-performance liquid chromatography (HPLC). Subsequently, the sequences of purified peptides were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). Molecular docking was used to analyze the interaction of these peptides with the transforming growth factor-ß1 (TGF-ß1) receptor. Two (GPPGVPGPGPL, TSLPVPAPV) of these novel peptides displayed lower binding energies to the TGF-ß1 receptor (-8.18 kcal mol-1, -8 kcal mol-1). Finally, the above two peptides were synthesized chemically and their in vitro anti-liver fibrosis activity was verified by MTT assay. Among them, GPPGVPGPGPL showed a better in vitro anti-liver fibrosis activity (IC50: 80.13 µM). We established a method to obtain anti-liver fibrosis peptides from turtle shells by using bioactivity-guided isolation with molecular docking. Turtle shell protein is an excellent source of anti-liver fibrosis peptides which can offer therapeutic and commercial benefits as an ingredient in functional foods.

Angelica Yinzi alleviates 1-chloro-2,4-dinitrobenzene-induced atopic dermatitis by inhibiting activation of NLRP3 inflammasome and down-regulating the MAPKs/NF-kB signaling pathway.

Background: Atopic dermatitis (AD), characterized by eczema as a chronic pruritic inflammatory skin disease, has become a serious health problem with recurrent clinical episodes. However, current clinical treatments have limited relief and are accompanied by adverse effects. Therefore, there is a necessity to develop new effective drugs for AD treatment. Angelica Yinzi (AYZ) is a classic ancient prescription for nourishing blood, moistening dryness, dispelling wind, and relieving itching. However, its mechanism for alleviating atopic dermatitis remains unknown. Therefore, this study aimed at determining the effects of AYZ and its potential mechanism in alleviating AD-like symptoms. Methods: In the present study, we used 1-chloro-2,4-dinitrobenzene (DNCB) to establish a mouse model of atopic dermatitis, where DNCB readily penetrates the epidermis to cause inflammation. Histopathological analysis was performed to examine the thickening of dorsal skin and infiltration in the inflammatory and mast cells in C57BL/6 mice. Additionally, the immunoglobulin E (IgE) levels in serum were determined by enzyme-linked immunosorbent assay (ELISA) kits. The IL-1ß and TNF-α expression were detected using qRT-PCR. Next, the Western blotting and immunohistochemistry assays were performed to assess the contribution of MAPKs/NF-κB signaling pathways and the NLRP3 inflammasome in AD responses. Results: Histopathological examination revealed that AYZ reduced the epidermal thickness of AD-like lesioned skin and repressed the infiltration of mast cells into AD-like lesioned skin. AYZ significantly decreased the phosphorylation of p38 MAPK, JNK, ERK and NF-κB and downregulated serum IgE levels and IL-1ß and TNF-α mRNA levels. Additionally, the NLRP3, ASC, Caspase-1, and IL-1ß expression in dorsal skin were effectively down-regulated following AYZ treatment (p < 0.05 and p < 0.01). Conclusion: These findings revealed that AYZ effectively suppressed AD-induced skin inflammation by inhibiting the activation of the NLRP3 inflammasome and the MAPKs/NF-kB signaling. Therefore, AYZ is a potential therapeutic agent against AD in the clinical setting.

Opportunities and challenges of patient-derived models in cancer research: patient-derived xenografts, patient-derived organoid and patient-derived cells.

BACKGROUND: As reported, preclinical animal models differ greatly from the human body. The evaluation model may be the colossal obstacle for scientific research and anticancer drug development. Therefore, it is essential to propose efficient evaluation systems similar to clinical practice for cancer research. MAIN BODY: While it has emerged for decades, the development of patient-derived xenografts, patient-derived organoid and patient-derived cell used to be limited. As the requirements for anticancer drug evaluation increases, patient-derived models developed rapidly recently, which is widely applied in basic research, drug development, and clinical application and achieved remarkable progress. However, there still lack systematic comparison and summarize reports for patient-derived models. In the current review, the development, applications, strengths, and challenges of patient-derived models in cancer research were characterized. CONCLUSION: Patient-derived models are an indispensable approach for cancer research and human health.

Extracts of Poria cocos improve functional dyspepsia via regulating brain-gut peptides, immunity and repairing of gastrointestinal mucosa.

BACKGROUND: Poria cocos (Schw.) Wolf (PC), a fungus, has been used for more than 2000 years as a food and medicine in China. It has a very good therapeutic effect for functional dyspepsia (FD). However, the material basis and mechanism of PC on FD were not reported. PURPOSE: To investigate the function and potential mechanisms of PC including its three extracts (triterpenoid, PCT; water-soluble polysaccharide, PCWP; acidic polysaccharide, PCAP) on FD. STUDY DESIGN: The study explored the therapeutic effect of PC and its three extracts on FD in rats for the first time and discussed its mechanisms based on brain-gut peptides, immunity and repair of the gastrointestinal mucosa. METHODS: The chemical components of PC extracts were analyzed and quantified using ultra high performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS) and gel permeation chromatography coupled with size exclusion chromatography (GPC/SEC). The FD rat models were established using weight-loaded forced swimming and alternate-day fasting for 42 days. After 14 days of treatment, the effect and mechanisms were investigated using ELISA, histopathology, immunohistochemistry as well as Western blot. RESULTS: Seventy-seven triterpenoids in PCT were identified. PCWP was primarily composed of component A (Mw: 3.831 × 107 Da), component B (Mw: 5.650 × 106 Da) and component C (Mw: 113,117 Da). PCAP was a homogeneous composition with an average Mw of 74,320 Da. PCT, PCWP and PCAP alleviated the symptoms of FD. These extracts promoted the repair of gastrointestinal mucosa and regulated the balance between the T helper cell (Th)1/Th2 axis and the Th17/Treg axis. PCT and PCWP regulated brain-gut peptides more effectively, PCWP and PCAP enhanced immunity more effectively. Further study demonstrated that these extracts may have enhanced immunity via the Toll-like receptor (TLR) and c-Jun N-terminal kinase (JNK) signaling pathways. CONCLUSIONS: PC extracts showed therapeutic effects on FD rats, and the mechanism of action involved multiple pathways. PCAP, which is often discarded in traditional applications, was effective. Our study provides new ideas for the application and development of PC extracts.

MicroRNA-483-5p Predicts Poor Prognosis and Promotes Cancer Metastasis by Targeting EGR3 in Nasopharyngeal Carcinoma.

BACKGROUND: MicroRNAs, as small non-coding RNAs, play an important role in tumorigenesis. MiR-483-5p was found to have a significant increase as a diagnostic biomarker of nasopharyngeal carcinoma (NPC), not only in plasma from NPC patients but also in tumor cell lines and biopsy tissues in our previous study. However, its function and mechanism in NPC are still unclear. METHODS: Tissue microarray including 178 primary NPC and 35 adjacent non-cancerous nasopharyngeal mucosal tissues was used to further validate the overexpression of miR-483-5p. Wound healing and invasion assays were conducted to verify its biological function. RNA sequencing (RNA-seq) and dual-luciferase reporter assay was performed to explore its target, and it was verified in fresh biopsy tissues from 23 NPC patients and 9 patients with chronic nasopharyngitis. RESULTS: MiR-483-5p was highly expressed in NPC tissues than in adjacent non-cancerous tissues. It was found to have a significant correlation with poor overall survival (OS) [hazard ratio (HR) = 2.89, 95% confidence interval (CI) = 1.00-8.35, p = 0.041] and progression-free survival (PFS) (HR = 1.95, 95%CI = 1.06-3.60, p = 0.029) of NPC patients. Silencing of its expression inhibited the migratory and invasive capacities of NPC cells in vitro. EGR3 (early growth response 3) was identified as a direct target, and inhibiting miR-483-5p expression markedly enhanced the expression of EGR3 at both the mRNA and protein levels. Besides, a significant decrease of EGR3 expression was found in fresh biopsy tissues from NPC patients, in contrast to miR-483-5p expression. Furthermore, directly decreasing the expression of EGR3 could enhance the migration and invasion of NPC cells. CONCLUSION: The newly identified miR-483-5p/EGR3 pathway provides further insights into the development and metastasis of NPC and may provide a potential therapeutic target for NPC treatment in order to improve survival of NPC patients.

The Anti-Inflammatory Effect of Smilax china L. Extract on LPS-Stimulated THP-1 via Downregulation of MAPK and NF-κB Signaling Pathway.

BACKGROUND: Traditional Chinese medicine Smilax is the rhizome of liliaceous plant Smilax china L., which is used to treat pelvic inflammatory disease and anxieties. PURPOSE: To investigate the mechanism of anti-inflammatory activity of the extract from Smilax china L. (ES). METHODS: The components of ES were identified by UPLC-QTOF-MS/MS. The anti-inflammatory activities were evaluated in xylene-induced ear oedema and egg white-induced plantar swelling test. Cell viability was examined by CCK-8 assay. The inflammatory mediators, proinflammatory cytokines, and MAPK and NF-κB signals in LPS-stimulated THP-1 cells were determined using ELISA, real-time PCR, and Western blot, respectively. RESULTS: 20 compounds of ES were confirmed by comparing with the reference substance. ES displayed more prominent anti-inflammatory activity than the positive control "Jin Gang Teng" capsule in the in vivo acute inflammatory model. ES suppressed the expression of PGE2 and 6-Keot-PGF1 α, and the ratio of IC50 (COX-1)/IC50 (COX-2) of ES was 3.15, which indicated that ES could selectively inhibit COX-2. ES dose-dependently (12.5, 25, and 50 mg/L) decreased the production and mRNA levels of proinflammatory cytokines IL-1ß, IL-6, and TNF-α. Furthermore, ES significantly decreased LPS-induced phosphorylation of p38, JNK, ERK1/2, and p65, inhibiting the expression of IKKα and the degradation of IκBα. CONCLUSION: The results suggested that ES could selectively inhibit the activity of COX-2, and the anti-inflammatory effect of ES was associated with the inhibition of IL-1ß, IL-6, and TNF-α via negative regulation of MAPK and NF-κB signaling pathways in LPS-induced THP-1 cells.

MicroRNAs in Body Fluids: A More Promising Biomarker for Clear Cell Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is the second most common cancer of the urinary system, accounting for approximately 10-15% of kidney cancers in the world. Clear cell renal cell carcinoma (ccRCC) is the most common RCC subtype with the highest mortality. Surgical resection or puncture of tumor tissue is still an important clinical treatment and diagnosis of ccRCC, but its high recurrence rate and poor prognosis often lead to the short survival period of patients. Hence, the development of novel molecular biomarkers is of great clinical importance. miRNAs are endogenous non-coding small RNAs with a length of 19-24 nt. A growing number of studies have reported that miRNAs, as proto-oncogenes or tumor suppressor genes, play a key role in the development of ccRCC and might be effective diagnostic and prognostic biomarkers. In addition, miRNAs can also predict the efficacy of treatment drug, thus improving the accuracy of clinical medication. Furthermore, non-invasive detection of miRNAs or extracellular vesicles (EV) in body fluids has better convenience and repeatability, which shows remarkable advantages compared with tissue detection. In this review, we summarized the typical miRNAs reported in recent years and place emphasis on evaluating miRNAs in different body fluids to provide reference for the clinical diagnosis and prognosis of ccRCC in the future.

Huganbuzure Granule Attenuates Concanavalin-A-Induced Immune Liver Injury in Mice via Regulating the Balance of Th1/Th2/Th17/Treg Cells and Inhibiting Apoptosis.

In Uygur medicine, Huganbuzure granule (HBG) is one of the classical prescriptions for liver protection. However, its role in immune liver injury remains unknown. This study evaluates the effect of HBG on concanavalin-A- (ConA-) induced immune liver injury and investigates its protective underlying mechanism. BALB/c mice were randomly divided into five groups (n = 24 mice per group): control, ConA, 1.6 g/kg HBG + ConA, 3.2 g/kg HBG + ConA, and 6 mg/kg prednisolone + ConA. HBG was intragastrically administrated once daily for ten consecutive days, prior to ConA (20 mg/kg) injection. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), superoxide dismutase (SOD), and malondialdehyde (MDA) in mouse serum were measured after ConA injection. Moreover, liver-related mRNA levels were evaluated by qPCR. The detection of liver-related proteins was assessed by immunohistochemistry and western blot analysis. Compared with the ConA group, HBG reduced the mRNA expression of IL-17A and IFN-γ and the protein expression of T-bet and ROR-γt. In addition, HBG increased the mRNA expression of IL-4 and TGF-ß and protein expression of GATA3 and Foxp3, indicating that HBG regulated the balance of Th1/Th2 and Th17/Treg. Furthermore, HBG alleviated immune liver injury by reducing oxidative stress, inhibiting apoptosis, and decreasing the expression of p-JNK, p-ERK, p-p38, p-JAK1, p-STAT1, p-STAT3, and IRF1. Our data suggested that HBG attenuated ConA-induced immune liver injury by regulating the immune balance and inhibiting JAK1/STATs/IRF1 signaling, thereby reducing apoptosis induced by JNK activation. The findings indicate that HBG may be a promising drug for immune liver injury.

Network pharmacology and molecular docking reveal the mechanism of Scopoletin against non-small cell lung cancer.

AIMS: Scopoletin is a natural anticarcinogenic and antiviral coumarin component. Many studies have proved its anti-cancer effect, and after the preliminary screening of this study, Scopoletin had the best inhibitory effect on Non-small cell lung cancer (NSCLC). But its mechanism for treating NSCLC is still unclear. Therefore, network pharmacology and molecular docking technology were used to explore the potential anti-NSCLC targets and pathways of Scopoletin. The results were verified in vitro. MAIN METHODS: First, Scopoletin was isolated from Fennel and screened to conduct cell proliferation assay on Human lung cancer cell line A549, Human colon cancer cell line HCT-116 and Human hepatoma cell line HepG2 respectively, through the MTT test. Then, the key targets and related pathways were screened through Protein-protein Interaction (PPI) network and "component-target-pathway" (C-TP) network constructed by network pharmacology. And the key targets were selected to dock with Scopoletin via molecular docking. A549 and Human normal lung epithelial cell BEAS-2B were used to verify the results, finally. KEY FINDINGS: Through MTT, A549 was chosen as the test cancer cell. From network pharmacology, 16 targets, 27 signaling pathways and 16 GO items were obtained (P < 0.05). The results of PPI network and molecular docking showed that EGFR, BRAF and AKT1 were the key targets of Scopoletin against NSCLC, which were consistent with the western-blot results. SIGNIFICANCE: Through network pharmacology, molecular docking and experiments in vitro, Scopoletin was verified to against NSCLC through RAS-RAF-MEK-ERK pathway and PI3K/AKT pathway.

Yu Gan Long Ameliorates Hepatic Fibrosis by Inhibiting PI3K/AKT, Ras/ERK and JAK1/STAT3 Signaling Pathways in CCl4-induced Liver Fibrosis Rats.

Yu Gan Long (YGL) is a Chinese traditional herbal formula which has been reported to attenuate liver fibrosis for many years and we have explored its anti-fibrotic mechanism through blocking transforming growth factor (TGF-ß) in the previous study. But the mechanisms associated with platelet-derived growth factor (PDGF)-BB remain obscure. In this study, we further investigated the mechanism of YGL reducing carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Our results showed that YGL suppressed CCl4-induced upregulation of collagen IV (Col IV), type HI precollagen (PCHI), hyaluronuc acid (HA) and laminin (LN), which are implicated in liver fibrosis. Also, YGL reduced the α-smooth muscle actin (α-SMA) expression, which acts as the indicator of liver fibrosis. Furthermore, YGL decreased the serum levels of hepatic stellate cell (HSC) mitogen PDGF-BB and inflammation cytokines, including TNF-α, IL-1ß, IL-6. Markers involved in liver fibrosis, such as Ras, p-Raf-1, p-ERK1/2, p-JNK, p-P38, p-PI3K, p-AKT, p-JAKl, p-STAT3 were downregulated significantly after treatment with YGL. Our results indicated that YGL ameliorated CCl4-induced liver fibrosis by reducing inflammation cytokines production, and suppressing Ras/ERK, PI3K/AKT, and JAK1/STAT3 signaling pathways, which provided further evidence towards elucidation of the anti-fibrotic mechanism of YGL.

Deciphering the Pharmacological Mechanisms of Taohe-Chengqi Decoction Extract Against Renal Fibrosis Through Integrating Network Pharmacology and Experimental Validation In Vitro and In Vivo.

Taohe-Chengqi decoction (THCQ), a classical traditional Chinese medicinal (TCM) formula, has been extensively used for treating chronic kidney disease (CKD). However, the biological activity and mechanisms of action of its constituents against renal fibrosis have not yet been investigated thoroughly. This study was aimed at devising an integrated strategy for investigating the bioactivity constituents and possible pharmacological mechanisms of the n-butanol extract of THCQ (NE-THCQ) against renal fibrosis. The n-butanol extract of THCQ was prepared by the solvent extraction method. The components of NE-THCQ were analyzed using UPLC-Q/TOF-MS/MS techniques and applied for screening the active components of NE-THCQ according to their oral bioavailability and drug-likeness index. Then, we speculated the potential molecular mechanisms of NE-THCQ against renal fibrosis through pharmacological network analysis. Based on data mining techniques and topological parameters, gene ontology, and pathway enrichment, we established compound-target (C-T), protein-protein interaction (PPI) and compound-target-pathway (C-T-P) networks by Cytoscape to identify the hub targets and pathways. Finally, the potential molecular mechanisms of NE-THCQ against renal fibrosis, as predicted by the network pharmacology analyses, were validated experimentally in renal tubular epithelial cells (HK-2) in vitro and against unilateral ureteral obstruction models in the rat in vivo. We identified 26 components in NE-THCQ and screened seven bioactive ingredients. A total of 118 consensus potential targets associated with renal fibrosis were identified by the network pharmacology approach. The experimental validation results demonstrated that NE-THCQ might inhibit the inflammatory processes, reduce ECM deposition and reverse EMT via PI3K/AKT/mTOR and HIF-1α/VEGF signaling pathways to exert its effect against renal fibrosis. This study identified the potential ingredients of the NE-THCQ by UPLC-Q/TOF-MS/MS and explained the possible mechanisms of NE-THCQ against renal fibrosis by integrating network pharmacology and experimental validation.

Esculetin Inhibits Cancer Cell Glycolysis by Binding Tumor PGK2, GPD2, and GPI.

Glycolysis can improve the tolerance of tissue cells to hypoxia, and its intermediates provide raw materials for the synthesis and metabolism of the tumor cells. If it can inhibit the activity of glycolysis-related enzymes and control the energy metabolism of tumor, it can be targeted for the treatment of malignant tumor. The target proteins phosphoglycerate kinase 2 (PGK2), glycerol-3-phosphate dehydrogenase (GPD2), and glucose-6-phosphate isomerase (GPI) were screened by combining transcriptome, proteomics, and reverse docking. We detected the binding constant of the active compound using microscale thermophoresis (MST). It was found that esculetin bound well with three potential target proteins. Esculetin significantly inhibited the rate of glycolysis, manifested by differences of cellular lactate production and glucose consumption in HepG2 cells with or without esculetin. It was found that GPD2 bound strongly to GPI, revealing the direct interaction between the two glycolysis-related proteins. Animal tests have further demonstrated that esculetin may have anticancer effects by affecting the activity of PGK2, GPD2, and GPI. The results of this study demonstrated that esculetin can affect the glucose metabolism by binding to glycolytic proteins, thus playing an anti-tumor role, and these proteins which have direct interactions are potential novel targets for tumor treatment by esculetin.

A Potential Anti-cancer Compound Separated from the Chloroform Extract of the Chinese Medicine Formula Shenqi San.

This study examined anti-cancer compounds present in the chloroform extract of the Chinese medicine formula Shenqi San (CE-SS). Silica gel column chromatography, Sephadex LH-20, octadecylsilyl (ODS) column chromatography, and high performance liquid chromatography (HPLC) were used to separate the compounds from CE-SS. The structural formulas of the separated compounds were determined using 1D 1H and 13C experiments as well as high resolution electrospray ionization mass spectroscopy (HRESIMS). The corresponding results were compared with the reported literature data. A total of six compounds were separated and their structures were identified on the basis of corresponding spectroscopic and physico-chemical properties. They were Saikogenin F (I), Prosaikogenin D (II), Prosaikogenin F (III), ß-sitosterol (IV), 3ß,16ß,23-trihydroxy-13,28-epoxyurs-11-ene-3-O-ß-D-glucopyranoside (V), and methyl ursolic acid (VI). The separated compounds were evaluated in vitro for their inhibitory ability against the proliferation of A549 cells via MTT assay. Apoptosis was investigated using Annexin V-FITC/propidium iodide (PI) by flow cytometry. Apoptosis-associated proteins were examined by Western blotting. All the compounds were observed to have inhibitory activities against the proliferation of A549 cells to different degrees. Flow cytometry showed that compound V increased the proportion of apoptotic A549 cells in a dose-dependent manner. Western blotting showed that compound V increased the expression of Bax, cleaved-caspase-3, cleaved-caspase-9 and cleaved-poly ADP-ribose polymerase (PARP), and decreased the expression of Bcl-2. These results indicated that compound V featured a significant inhibitory effect on A549 cells when compared with other compounds, and it may be considered a potential drug against cancers.

Bioactive ingredients obtained from Cortex Fraxini impair interactions between FAS and GPI.

The high expression of fatty acid synthase (FAS) in tumor cells is consistent with their elevated requirement for fatty acids for cell membrane synthesis and energy supply to support their almost unlimited proliferation. The expression levels of FAS in tumor cells are related to their proliferation, invasion, and metastasis. This study investigated the possible bioactive ingredients (fraxin, esculetin, scopolin et al.) of Cortex Fraxini and their effects on the interaction between specific proteins. We used microscale thermophoresis (MST) to show that our target protein, FAS (screened by combining transcriptome and network pharmacology), bound to the active compounds in Cortex Fraxini. It was found that FAS bound strongly to Glucose-6-phosphate isomerase (GPI), and that scopolin could affect this interaction by proteomics and MST. The results of this study demonstrate that the active compounds in Cortex Fraxini could play an anti-tumor role by binding to FAS and inhibiting the interactions between FAS and GPI to affect glucose and lipid metabolism, and that the protein pathway is a potential novel target for tumor treatment.