Surface Plasmon Resonance-Based Membrane Protein-Targeted Active Ingredients Recognition Strategy: Construction and Implementation in Ligand Screening from Herbal Medicines.

Membrane proteins (MPs) are playing important roles in several biological processes. Screening new candidate compounds targeting MPs is important for drug discovery. However, it remains challenging to characterize the interactions between MPs and small-molecule ligands in a label-free method. In this study, a surface plasmon resonance (SPR)-based membrane protein-targeted active ingredients recognition strategy was constructed. This strategy contains two major modules: affinity detection module and ligand screening module. Through the combination of these two functional modules, it is feasible to screen small molecular ligands targeting MPs from herbal medicines. First, we have constructed high/low comparative C-X-C chemokine receptor type 4 (CXCR4)-expressed lentiviral particles (LVPs) models and characterized the expression levels. Then we immobilized LVPs on CM5 chips and detected the affinity between AMD3100 and CXCR4 by using affinity detection module. The KD of AMD3100 was 32.48 ± 3.17 nM. Furthermore, the suitability and robustness of the ligand screening module were validated by using AMD3100 as a positive compound. Subsequently, this module was applied in the screening of CXCR4 small molecular ligands from herbal medicine extracts. Senkyunolide I was screened out from Chuanxiong extract. The affinity constant between senkyunolide I and CXCR4 was 2.94 ± 0.36 µM. The Boyden chamber assay revealed that senkyunolide I could inhibit cell migration process. In conclusion, an SPR-based small molecular ligand recognition strategy combined with virus-based membrane protein stabilization method was constructed. The SPR-based membrane protein-targeted active ingredients recognition strategy will be an effective tool to screen target components from complex systems acting on MPs.

Bioactive compound reveals a novel function for ribosomal protein S5 in hepatic stellate cell activation and hepatic fibrosis.

UNLABELLED: Liver fibrosis and its endstage, cirrhosis, represent a major public health problem worldwide. Activation of hepatic stellate cells (HSCs) is a central event in hepatic fibrosis. However, the proteins that control HSC activation are incompletely understood. Here we show that (6aS, 10S, 11aR, 11bR, 11cS)-10-methylamino-dodecahydro-3a, 7a-diaza-benzo [de]anthracene-8-thione (MASM) exhibits potent inhibitory activity against liver fibrosis in vitro and in vivo associated with the reduction of Akt phosphorylation. Furthermore, ribosomal protein S5 (RPS5) was identified as a direct target of MASM, which stabilized RPS5 in cultured HSCs and in the liver of experimental animals after dimethylnitrosamine (DMN) or bile duct ligation (BDL). Functional studies revealed that RPS5 could prevent HSC activation. RPS5 overexpression in HSCs resulted in Akt dephosphorylation at both Ser473 and Thr308, and led to subsequent dephosphorylation of GSK3ß or P70S6K. Progression of DMN- and BDL-induced hepatic fibrosis was aggravated by Rps5 knockdown and alleviated by RPS5 overexpression, which correlated with the modulation of Akt phosphorylation and HSC number in the fibrotic livers. Moreover, RPS5 was substantially reduced in the transdifferentiated HSCs, experimental fibrotic livers, and human cirrhosis samples. CONCLUSION: These results demonstrate that RPS5 is implicated in hepatic fibrogenesis and may represent a promising target for potential therapeutic intervention in liver fibrotic diseases.

Antifibrotic effects of luteolin on hepatic stellate cells and liver fibrosis by targeting AKT/mTOR/p70S6K and TGFß/Smad signalling pathways.

BACKGROUND & AIMS: Luteolin has been reported to exert antifibrogenic effects in CCl4 -induced hepatic fibrosis in mice. However, limited information is available on the cellular and molecular events responsible for this effect. This study focused on the action of luteolin on hepatic stellate cells (HSCs) and the relevant signalling molecules and pathways as well as the antifibrotic efficacy in multiple models of fibrosis. METHODS: The in vitro effect of luteolin on rat HSCs and HSC-T6 cells was assessed using proliferation assays, invasion chamber, quantitative real-time PCR analysis and Western blotting. The in vivo effect of luteolin on progression of fibrosis was assessed in three experimental rat models induced by CCl4 , dimethylnitrosamine (DMN) and bile duct ligation (BDL). RESULTS: Luteolin inhibited proliferation, migration, collagen synthesis as well as expression of fibrosis-related genes in the activated HSCs and HSC-T6 cells stimulated with or without transforming growth factor-ß1(TGFß1) or platelet-derived growth factor (PDGF). Luteolin induced HSC apoptosis associated with the increased caspase 3 activity and p53 expression, and induced G1 arrest with the decreased expression of bcl-2, Cyclin E and p-Cdk-2. Moreover, luteolin significantly inhibited PDGF and TGFß1-simulated phosphorylation of AKT and Smad pathway. In vivo study showed that luteolin administration markedly alleviated hepatic fibrosis along with reduced elevations of alanine aminotransferase and aspartate aminotransferase. HSCs were found to undergo apoptosis and decreased expression of p-Smad2 and p-AKT in luteolin-treated animals. CONCLUSIONS: This study demonstrates that luteolin prevents the progression of liver fibrosis through multiple mechanisms and indicates that luteolin has potential for effective treatment of liver fibrosis.

Salvianolic acid B inhibits hepatic stellate cell activation and liver fibrosis by targeting PDGFRß.

Liver fibrosis is a reversible pathological process and a wound healing response to liver injury. As an early stage of various liver diseases, liver fibrosis can develop into cirrhosis, liver failure, and even liver cancer if not controlled in time. Salvia miltiorrhiza is a medicinal plant with hepatoprotective effects. Salvianolic acid B (Sal B) is the representative component of S. miltiorrhiza. Many studies have reported the anti-liver fibrosis effects and mechanisms of Sal B. However, the direct anti-fibrotic targets of Sal B have not yet been reported. Platelet-derived growth factor receptor ß (PDGFRß) is one of the most classical targets in liver fibrosis, which is closely related to hepatic stellate cells (HSCs) activated. Previously, we established and applied a PDGFRß affinity chromatography model, and found that Sal B binds well to PDGFRß. Therefore, this study aimed to investigate the direct targets of Sal B against liver fibrosis. We confirmed the binding ability of Sal B to PDGFRß by molecular docking and a surface plasmon resonance biosensor. Our findings indicated that Sal B targeted PDGFRß to inhibit the activation, migration and proliferation of HSCs and suppressed the PDGF-BB-induced PDGFRß signaling pathway. Annexin V-FITC/PI assay showed that Sal B reversed the PDGF-BB-induced decrease in HSC apoptosis rate. In the mouse liver fibrosis model, Sal B inhibited the PDGFRß signaling pathway, HSC activation and reduced inflammatory response, ultimately improved CCl4-induced liver fibrosis. In summary, the direct anti-fibrotic targets of Sal B may be PDGFRß, and this study clarified the anti-liver fibrosis effects and mechanism of Sal B.

Salvianolic acid B suppresses hepatic stellate cell activation and liver fibrosis by inhibiting the NF-κB signaling pathway via miR-6499-3p/LncRNA-ROR.

BACKGROUND: Long non-coding RNA (LncRNAs) have been reported to play an important role in liver fibrosis and are closely associated with hepatic stellate cell (HSC) activation. We previously found that salvianolic acid B (Sal B) improves liver fibrosis by regulating the NF-κB signaling pathway. However, whether the LncRNA, regulator of reprogramming (LncRNA-ROR) plays a role in Sal B-mediated anti-fibrosis effects via the NF-κB signaling pathway remain unclear. PURPOSE: This study aimed to evaluate the effects of Sal B on HSC activation and liver fibrosis and investigate its mechanism from the perspective of LncRNA-ROR-mediated NF-κB signaling pathways. METHODS: LX-2 and T6 cell lines were cultured. Animal models of liver fibrosis were established using CCl4 in male BALB/c mice. Primary HSCs were isolated from mice and cultured. Serum biochemical and liver histological analyses were performed to evaluate the effects of Sal B on liver fibrosis. The index of HSC activation and the expression of LncRNA-ROR, microRNAs (miRNAs), and inflammatory factors were determined by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) or immunofluorescence staining. Cell proliferation was measured by a Cell Counting Kit-8 (CCK-8). NF-κB signaling-associated protein levels were assessed using western blotting or immunofluorescence staining. A luciferase reporter assay was used to detect transcription activity. RESULTS: In this study, a lower level of LncRNA-ROR was found during Sal B attenuating HSC activation in HSCs. Mechanistically, Sal B impeded the NF-κB signaling pathway to inhibit HSC proliferation and activation by downregulating LncRNA-ROR. Additionally, Sal B upregulated miR-6499-3p to target LncRNA-ROR for degradation. Functionally, Sal B treatment ameliorated CCl4-induced liver fibrosis in mice by inhibiting HSC activation. CONCLUSION: Sal B suppresses HSC activation and liver fibrosis via regulation of miR-6499-3p/LncRNA-ROR-mediated NF-κB signaling pathway. These results reveal a new molecular mechanism of Sal B on liver fibrosis from the insight of LncRNAs.

Neferine Exerts Antioxidant and Anti-Inflammatory Effects on Carbon Tetrachloride-Induced Liver Fibrosis by Inhibiting the MAPK and NF-κB/IκBα Pathways.

Reversible liver fibrosis is the consequence of diverse liver injuries. Oxidative stress combined with inflammation is the primary cause of carbon tetrachloride- (CCl4-) induced liver fibrosis. Neferine is a bibenzyl isoquinoline alkaloid, which has strong anti-inflammatory and antioxidant properties. The present study attempted to find its antiliver fibrosis effect and explore the potential mechanism to relieve oxidative stress and inflammation in rats with CCl4-induced liver fibrosis. Herein, we found that neferine noticeably mitigated fibrosis and improved liver function. Furthermore, neferine increased the activity of antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and catalase (CAT), but decreased the level of malondialdehyde (MDA). Neferine also decreased the levels of alpha-smooth muscle actin (α-SMA), transforming growth factor ß1 (TGF-ß1), and inflammatory factors. These results may demonstrate that neferine could effectively inhibit oxidative stress and inflammation in liver fibrosis. To account for the potential mechanism by which neferine relieves oxidative stress and inflammation in liver fibrosis rats, immunohistochemistry analyses and western blotting were performed. The results showed that neferine inhibited the mitogen-activated protein kinase (MAPK) pathway, as evidenced by the reduced phosphorylation of p38 MAPK, ERK 1/2, and JNK. And it inhibited the nuclear factor- (NF-) κB/IκBα pathway, as evidenced by preventing the translocation of NF-κB into nuclei. Our findings indicated a protective role for neferine, acting as an antioxidant and anti-inflammatory agent in CCl4-induced liver fibrosis.

Comparative two-dimensional GPC3 overexpressing SK-Hep1 cell membrane chromatography /C18/ time-of-flight mass spectrometry for screening selective GPC3 inhibitor components from Scutellariae Radix.

Screening active components targeting membrane proteins is important for drug discovery from traditional Chinese medicine. Cell membrane chromatography (CMC) has achieved a wide application in screening active components on pathological cells due to its high sensitivity and effectiveness. However, it is hard to clarify the specific target protein through simply using pathological and normal cells. In this study, a novel comparative two-dimensional (2D) cell membrane chromatography system was established. Based on the construction of hepatocellular carcinoma cell line SK-Hep1-GPC3 with high expression of protein Glypican-3 (GPC3), SK-Hep1-GPC3/CMC column was loaded to screen selective antitumor components from Scutellariae Radix according to the retention behaviors on column. Viscidulin I was retained on SK-Hep1-GPC3/CMC column, and showed 4.33 µM affinity to GPC3 according to surface plasmon resonance (SPR). The IC50 of viscidulin I on SK-Hep1-GPC3 cells was 18.01 µM in cell proliferation assay. Thus, this method can be applied to screen complex herbal medicines for ligands bound to specific target protein receptor related to hepatic carcinoma.

Synthesis and in vitro inhibitory activity of matrine derivatives towards pro-inflammatory cytokines.

Matrine, a sophora alkaloid, exhibited good anti-inflammation effects in our previous report. In the present study, a series of matrine derivatives were synthesized via classical Michael addition. Biological studies showed that the synthetic derivatives had good inhibitory effect towards TNF-α production and NFκB transcriptional activity. The introduction of various amino groups to the keto beta position could improve the biochemical profile, resulting in the identification of more potent derivates, such as 1f, with higher inhibitory activity than both matrine and sophoramine.

Development of 3-mercaptopropyltrimethoxysilane (MPTS)-modified bone marrow mononuclear cell membrane chromatography for screening anti-osteoporosis components from Scutellariae Radix.

Osteoporosis is a bone metabolic disease caused by the imbalance between osteoblasts and osteoclasts due to excess osteoclastogenesis, manifesting in the decrease of bone density and bone strength. Scutellariae Radix shows good anti-osteoporosis activity, but the effective component is still unclear. Cell membrane chromatography (CMC) is a biological affinity chromatography with membrane immobilized on a silica carrier as the stationary phase. It can realize a dynamical simulation of interactions between drugs and receptors on cell membrane, which is suitable for screening active compounds from complex systems. In this study, the components of Scutellariae Radix with potential anti-osteoporosis activity through inhibiting the differentiation from bone marrow mononuclear cells (BMMCs) to osteoclast were screened by a BMMC/CMC analytical system. Firstly, a new 3-mercaptopropyltrimethoxysilane (MPTS)-modified BMMC/CMC stationary phase was developed to realize covalent binding with cell membrane fractions. By investigating the retention time (t R) of the positive drug, the life span of the MPTS-modified CMC columns was significantly improved from 3 to 12 days. Secondly, 6 components of Scutellariae Radix were screened to show affinity to membrane receptors on BMMCs by a two-dimensional BMMC/CMC-TOFMS analytical system. Among them, tectochrysin demonstrated the best anti-osteoporosis effect in vitro, which has never been reported. We found that tectochrysin could inhibit the differentiation of BMMCs into osteoclasts induced by receptor activator of nuclear factor-κΒ ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) in a concentration-dependent manner in vitro. In vivo, it significantly reduced the loss of bone trabeculae in ovariectomized mice, and decreased the level of C-terminal cross-linking telopeptides of type 1 collagen (CTX-1), tartrate-resistant acid phosphatase 5b (TRAP-5b), interleukin 6 (IL-6) in serum. In conclusion, tectochrysin serves as a potential candidate in the treatment of osteoporosis. The proposed two-dimensional MPTS-modified BMMC/CMC-TOFMS analytical system shows the advantages of long-life span and fast recognition ability, which is very suitable for infrequent cell lines.

Comparative two-dimensional HepG2 and L02/ cell membrane chromatography/ C18/ time-of-flight mass spectrometry for screening selective anti-hepatoma components from Scutellariae Radix.

Screening active components from Chinese traditional medicine is an effective approach to discover new drugs or active structures. Cell membrane chromatography (CMC), developed rapidly because of its high sensitivity and effectiveness, has achieved a wide application in screening active components on pathological cells or tissues. However, it is hard to clarify the selectivity between pathological and normal tissues through simply using pathological cells. In this study, a novel comparative two-dimensional (2D) cell membrane chromatography system was established. Briefly, hepatic carcinoma HepG2 CMC columns and normal hepatic L02 CMC columns were simultaneously loaded to screen potential selective antitumor components from Scutellariae Radix by comparing the retention behaviors on two kinds of cells. Totally 13 components in Scutellariae Radix retained on both HepG2/ CMC and L02/ CMC columns. Among them, three components, oroxylin A, wogonin and chrysin, were screened out to perform stronger affinity on HepG2 columns, and in further cell proliferation assay, IC50 of these three compounds of HepG2 cells were 9.66 µM, 66.77 µM and 36.26 µM respectively, while of L02 cells, IC50 of chrysin was 59.10 µM and over 200 µM of the other two components. On the whole, the toxity of these three compounds to hepatoma cells was stronger than to normal cells. It can be supposed that oroxylin A, wogonin, and chrysin own the potential to be developed as selective anti-hepatoma active components, which expects further research to validate.

Sophocarpine and matrine inhibit the production of TNF-alpha and IL-6 in murine macrophages and prevent cachexia-related symptoms induced by colon26 adenocarcinoma in mice.

The present study aims to access the effects of sophora alkaloids on the production of pro-inflammatory cytokines and evaluate their therapeutic efficiency on cachexia. The comparative study showed that all sophora alkaloids tested here, including matrine, oxymatrine, sophocarpine, sophoramine, and sophoridine, inhibited TNF-alpha and IL-6 production in both RAW264.7 cells and murine primary macrophages, and sophocarpine showed the most potent inhibitory effect among them. Quantification of TNF-alpha and IL-6 mRNA in RAW264.7 cells by real-time RT-PCR revealed that both sophocarpine and matrine suppressed TNF-alpha and IL-6 expression and sophocarpine has stronger suppressing potency than matrine. Inoculation (s.c.) of colon26 adenocarcinoma cells into BALB/c mice induced cachexia, as evidenced by progressive weight loss, reduction in food intake, wasting of gastrocnemius muscle and epididymal fat, and increase in serum levels of TNF-alpha and IL-6. Administration of 50 mg/kg/d sophocarpine or matrine for 5 days from the onset of cachexia did not inhibit the tumor growth but resulted in attenuation of cachexia symptoms. Furthermore, sophocarpine and matrine decreased the serum levels of TNF-alpha and IL-6, and sophocarpine showed a better therapeutic effect than matrine. These results suggest that sophocarpine and matrine exert anti-cachectic effects probably through inhibition of TNF-alpha and IL-6.

A method for screening active components from Chinese herbs by cell membrane chromatography-offline-high performance liquid chromatography/mass spectrometry and an online statistical tool for data processing.

Cell membrane chromatography (CMC) has been successfully applied to screen bioactive compounds from Chinese herbs for many years, and some offline and online two-dimensional (2D) CMC-high performance liquid chromatography (HPLC) hyphenated systems have been established to perform screening assays. However, the requirement of sample preparation steps for the second-dimensional analysis in offline systems and the need for an interface device and technical expertise in the online system limit their extensive use. In the present study, an offline 2D CMC-HPLC analysis combined with the XCMS (various forms of chromatography coupled to mass spectrometry) Online statistical tool for data processing was established. First, our previously reported online 2D screening system was used to analyze three Chinese herbs that were reported to have potential anti-inflammatory effects, and two binding components were identified. By contrast, the proposed offline 2D screening method with XCMS Online analysis was applied, and three more ingredients were discovered in addition to the two compounds revealed by the online system. Then, cross-validation of the three compounds was performed, and they were confirmed to be included in the online data as well, but were not identified there because of their low concentrations and lack of credible statistical approaches. Last, pharmacological experiments showed that these five ingredients could inhibit IL-6 release and IL-6 gene expression on LPS-induced RAW cells in a dose-dependent manner. Compared with previous 2D CMC screening systems, this newly developed offline 2D method needs no sample preparation steps for the second-dimensional analysis, and it is sensitive, efficient, and convenient. It will be applicable in identifying active components from Chinese herbs and practical in discovery of lead compounds derived from herbs.

Combination of comprehensive two-dimensional prostate cancer cell membrane chromatographic system and network pharmacology for characterizing membrane binding active components from Radix et Rhizoma Rhei and their targets.

Prostate cancer (PCa) is a common and fatal cancer for men and effective treatment options are still not enough for patients. Radix et Rhizoma Rhei had been applied to treat PCa long-termly and effectively when combined with surgical treatment and chemotherapy. However, its active components and target proteins are still not quite clear. As membrane receptors play a vital role in PCa, in this study, a novel strategy that combines comprehensive 2D 3-aminopropyltriethoxysilane-decorated prostate cancer cell (DU145) membrane chromatographic (CMC) system with network pharmacology approach was developed to characterize membrane binding active components proteins from Radix et Rhizoma Rhei and their targets. Thirteen active components were screened out by CMC system, among which emodin and rhapontigrnin with good membrane binding behaviors were validated to show ideal inhibitory effects on DU145 cells by cell viability and cell apoptosis assays. Five membrane proteins were predicted as the potential targets by the a specific network pharmacology approach, among which mast/stem cell growth factor receptor Kit (KIT) was identified as the most possible target by network data mining. Surface plasmon resonance analysis verified that the dissociation constant (KD) of rhapontigrnin and emodin with KIT was 6.06 × 10-5 M and 8.82 × 10-5 M, respectively. Our results showed that the combination of comprehensive 2D CMC system and network pharmacology based target identification could not only rapidly identify the membrane binding components but also find the potential membrane protein targets with high confidence, which could broaden the range of application scope of CMC, especially for the screening of active compounds from complex chemical samples using primary pathologic cell lines.

Antifibrotic effects of tanshinol in experimental hepatic fibrosis by targeting PI3K/AKT/mTOR/p70S6K1 signaling pathways.

BACKGROUND AND AIMS: Tanshinol is a water-soluble bioactive monomer purified from the dried root of Salvia miltiorrhiza and has been reported to exert hepatoprotective efficacy in rodents. However, detailed knowledge remains limited about tanshinol's effects on hepatic stellate cells (HSC) functions, which play an essential role in the progression of hepatic fibrosis. Our research primarily focused on the effects of tanshinol on activation and apoptosis of HSC and further investigated PI3K/AKT/mTOR/p70S6K1 signaling pathways' participation in the pathogenesis of hepatic fibrosis in carbon tetrachloride (CCl4)-induced hepatic fibrosis. METHODS: The antifibrotic effects of tanshinol on the development of fibrosis were established through CCl4-induced rat models. 48 male Sprague-Dawley (SD) rats were randomized to the normal group, CCl4 model group, and two tanshinol treatment groups, including a lower dosage group as well as a higher dosage group. RESULTS: Tanshinol prominently mitigated liver fibrosis and reduced levels of alanine aminotransferase (ALT), aspartate transaminase (AST), hydroxyproline content, and other serum markers of liver fibrosis. Concomitantly, tanshinol regulated the degradation of extracellular matrix as well as hepatic sinusoid and inhibited the expression of inflammation-related genes. Tanshinol promoted apoptosis of the activated HSC and increased cleaved caspase 3 levels and the number of TUNEL-positive HSC in two tanshinol-administered groups. In addition, tanshinol significantly inhibited the expression of phosphorylated AKT, phosphorylated mTOR, and phosphorylated p70S6K1 proteins. CONCLUSIONS: This study demonstrates that tanshinol exerts antifibrotic effects through targeting multiple mechanisms correlated with PI3K/AKT/mTOR/p70S6K1 signaling pathways, and has the prospect of becoming a new treatment strategy for hepatic fibrosis.

Ginsenoside Rb1 and paeoniflorin inhibit transient receptor potential vanilloid-1-activated IL-8 and PGE2 production in a human keratinocyte cell line HaCaT.

Ginsenoside Rb1 (GRb1) and paeoniflorin (PF) are active substances of Chinese traditional herbs and have been commonly used to treat skin inflammation diseases, but little is known about the mechanisms involved. In the present study, the effects of GRb1 and PF on the production of inflammatory mediators and the possible mechanisms of transient receptor potential vanilloid-1 (TRPV1) in these mediators induction were explored. It has been shown that GRb1 and PF inhibited the productions of IL-8 and PGE2 induced by capsaicin (CAP) in HaCaT cells and HEK 293T-TRPV1 cells (which were transgenic and overexpressed TRPV1) but had no effect on HEK 293T mock cells (p<0.05). Besides, CAP was able to induce calcium influx and nuclear factor kappa B(NF-κB) transcriptional activity in HaCaT cells and HEK 293T-TRPV1 cells, but had no effect on HEK 293T mock cells. Furthermore, GRb1 inhibited CAP-induced calcium influx and NF-κB transcriptional activity in both HaCaT cells and HEK 293T-TRPV1 cells. However, PF decreased CAP-induced calcium influx and NF-κB transcriptional activity only in HaCaT cells. This would suggest that GRb1 inhibits CAP-induced calcium influx and NF-κB activity through TRPV1 signal, while calcium influx and NF-κB activity might not be involved in the inhibitory effect of PF on TRPV1 signal. Furthermore, the inhibitory rates of GRb1 and PF on IL-8 and PGE2 production were higher than those caused by capsazepine, an antagonist of TRPV1, suggesting that GRb1 and PF have great potential in clinical treatment of skin diseases.