Co-catalpol alleviates fluoxetine-induced main toxicity: Involvement of ATF3/FSP1 signaling-mediated inhibition of ferroptosis.

BACKGROUND: Fluoxetine is often used as a well-known first-line antidepressant. However, it is accompanied with hepatogenic injury as its main organ toxicity, thereby limiting its application despite its superior efficacy. Fluoxetine is commonly traditionally used combined with some Chinese antidepressant prescriptions containing Rehmannia glutinosa (Dihuang) for depression therapy and hepatoprotection. Our previous experiments showed that co-Dihuang can alleviate fluoxetine-induced liver injury while efficiencies, and catalpol may be the key ingredient to characterize the toxicity-reducing and synergistic effects. However, whether co-catalpol can alleviate fluoxetine-induced liver injury and its toxicity-reducing mechanism remain unclear. PURPOSE: On the basis of the first recognition of the dose and duration at which pre-fluoxetine caused hepatic injury, co-catalpol's alleviation of fluoxetine-induced hepatic injury and its pathway was comprehensively elucidated. METHOD AND RESULTS: The hepatoprotection of co-catalpol was evaluated by serum biochemical indexes sensitive to hepatic injury and multiple staining techniques for hepatic pathologic analysis. Subsequently, the pathway by which catalpol alleviated fluoxetine-induced hepatic injury was predicted by network pharmacology to be predominantly the inhibition of ferroptosis. These were validated and confirmed in subsequent experiments with key technologies and diagnostic reagents related to ferroptosis. Further molecular docking showed that activating transcription factor 3 (ATF3) and ferroptosis suppressor protein 1 (FSP1) were the the most prospective molecules for catalpol and fluoxetine among many ferroptosis-related molecules. The critical role of ATF3/FSP1 signaling was further observed by surface plasmon resonance, diagnostic reagents, transmission electron microscopy, Western blot, real-time PCR, immunofluorescence, and immunohistochemistry. Results showed that fluoxetine directly bound to ATF3 and FSP1; agonisting ATF3 or blocking FSP1 abolished the alleviation of catalpol on fluoxetine-induced liver injury, and both exacerbated ferroptosis. Moreover, co-catalpol significantly enhanced the antidepressant efficacy of fluoxetine against depressive behaviours in mice. CONCLUSION: The hepatic impairment properties of fluoxetine were largely dependent on ATF3/FSP1 target-mediated ferroptosis. Co-catalpol alleviated fluoxetine-induced hepatic injury while enhancing its antidepressant efficacy, and that ATF3/FSP1 signaling-mediated inhibition of ferroptosis was involved in its co-administration detoxification mechanism. This study was the first to reveal the hepatotoxicity characteristics, targets, and mechanisms of fluoxetine; provide a detoxification and efficiency regimen by co-catalpol; and elucidate the detoxification mechanism.

Investigation of the principle of concoction by using the processing excipient Glycyrrhiza uralensis Fisch. juice to reduce the main toxicity of Dioscorea bulbifera L. and enhance its main efficacy as expectorant and cough suppressant.

ETHNOPHARMACOLOGICAL RELEVANCE: Dioscorea bulbifera L. (Rhizoma Dioscoreae Bulbiferae; RDB) is commonly used as an expectorant and cough suppressant herb but is accompanied by severe hepatotoxicity. Using the juice of auxiliary herbs (such as Glycyrrhiza uralensis Fisch. (Glycyrrhizae Radix et Rhizoma; GRR) juice) in concocting poisonous Chinese medicine is a conventional method to reduce toxicity or increase effects. Our previous study found that concoction with GRR juice provided a detoxifying effect against the major toxic hepatotoxicity induced by RDB, but the principle for the detoxification of the concoction is unknown to date. AIM OF THE STUDY: The principle of concoction was investigated by using the processing excipient GRR juice to reduce the major toxic hepatotoxicity of RDB, and the efficacy of RDB as an expectorant and cough suppressant was enhanced. MATERIALS AND METHODS: In this study, common factors (RDB:GRR ratio, concocted temperature, and concocted time) in the concoction process were used for the preparation of each RDB concocted with GRR juice by using an orthogonal experimental design. We measured the content of the main toxic compound diosbulbin B (DB) and serum biochemical indicators and performed pathological analysis in liver tissues of mice to determine the best detoxification process of RDB concocted with GRR juice. On this basis, the biological mechanisms of target organs were detected by Western blot and enzyme-linked immunosorbent assay at the inflammation and apoptosis levels. Further, the effects of RDB on expectorant and cough suppressant with GRR juice were evaluated by the conventional tests of phenol red expectorant and concentrated ammonia-induced cough. Lastly, the major compounds in the GRR juice introduced to RDB concoction were determined. RESULTS: RDB concocted with GRR juice significantly alleviated DB content, serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase levels, and improved liver pathological damages. The best detoxification process was achieved by using an RDB:GRR ratio of 100:20 at 120 °C for 20 min. Further, RDB concocted with GRR juice down-regulated the protein levels of nuclear factor kappa B (NF-κB), cyclooxygenase 2 (COX-2), and Bcl-2 related X protein (Bax) in the liver and enhanced the expectorant and cough suppressant effects of RDB. Finally, liquiritin (LQ) and glycyrrhizic acid (GA) in the GRR juice were introduced to the RDB concoction. CONCLUSION: Concoction with GRR juice not only effectively reduced the major toxic hepatotoxicity of RDB but also enhanced its main efficacy as an expectorant and cough suppressant, and that the rationale for the detoxification and/or potentiation of RDB was related to the reduction in the content of the main hepatotoxic compound, DB, the introduction of the hepatoprotective active compounds, LQ and GA, in the auxiliary GRR juice, as well as the inhibition of NF-κB/COX-2/Bax signaling-mediated inflammation and apoptosis.

[Gender differences in antidepressant effect of raw Rehmanniae Radix].

For the first time, this study evaluated the gender differences and mechanisms of the antidepressant effects of raw Rehmanniae Radix(RRR) based on the classic depression model with traditional Chinese medicine syndrome of Yin deficiency and internal heat. The depression model with Yin deficiency and internal heat was established by the widely recognized and applied method of thyroxine induction of the classic depression model with Yin deficiency and internal heat(chronic unpredictable mild stress). Male and female mice were simultaneously treated with RRR. The study analyzed indicators of nourishing Yin and clearing heat, conventional antidepressant efficacy test indicators, and important biomolecules reflecting the pathogenesis and prevention and treatment mechanisms of depression, and conducted a correlation analysis of antidepressant efficacy, Yin-nourishing and heat-clearing efficacy, and biological mechanism in different genders, thereby comprehensively assessing the antidepressant effects of RRR on depression of Yin deficiency and internal heat, as well as its gender differences and mechanisms. RRR exhibited antidepressant effects in both male and female mouse models, and its antidepressant efficacy showed gender differences, with a superior effect observed in females. Moreover, the effects of RRR on enhancing or improving hippocampal neuronal pathology, nucleus-positive areas, postsynaptic dense area protein 95, and synaptophysin protein expression were more significant in females than in males. In addition, RRR significantly reversed the abnormal upregulation of nuclear factor(NF)-κB/cyclooxygenase 2(COX2)/NOD-like receptor thermal protein domain associated protein 3(NLRP3) pathway proteins in the hippocampus of both male and female mouse models. The antidepressant effects of RRR were more pronounced in depression female mice with Yin deficiency and internal heat syndrome, possibly due to the improvement of neuronal damage and enhancement of neuroplasticity. The antidepressant mechanisms of RRR for depression with Yin deficiency and internal heat syndrome may be associated with the downregulation of the NF-κB/COX2/NLRP3 pathway to reduce neuronal damage and enhance neuroplasticity.

NIR/pH dual-responsive polysaccharide-encapsulated gold nanorods for enhanced chemo-photothermal therapy of breast cancer.

Combination of different therapy modalities with multiple tumoricidal mechanisms has emerged as a promising anticancer strategy. Herein, we reported an aldehyde/catechol-functionalized hyaluronic acid (DAHA) and hydroxyethyl chitosan (HECS) decorated gold nanorod (GNR) platform for combined chemo-photothermal therapy of breast cancer. The DAHA was synthesized by conjugating dopamine onto oxidized hyaluronic acid. The nanoplatform was prepared by successively modifying GNR with DAHA via Au-catechol bonds, conjugating DOX onto DAHA moiety through Schiff base linkage, and coating HECS interlayer for charge-reversal and hyaluronic acid corona for tumor cell targeting. The resulting nanoplatform GNR-HADOXCH exhibited acid-triggered surface charge-reversal and pH/NIR dual-responsive drug release behaviors. The nanoplatform could be efficiently internalized into MCF-7 breast cancer cells and displayed greater cancer cell killing than individual modalities. Therefore, polysaccharide decoration could ensure the co-delivery of GNR and DOX into cancer cells, and the developed GNR-HADOXCH holds great potential for breast cancer treatment.

A novel pH-sensitive targeting polysaccharide-gold nanorod conjugate for combined photothermal-chemotherapy of breast cancer.

Chemo-photothermal combination therapy is a promising strategy for cancer treatment. In this study, to achieve the combined photothermal-chemotherapy of breast cancer, a pH-sensitive oxidized hyaluronic acid-decorated dihydroxyphenyl/hydrazide bifunctionalized hydroxyethyl chitosan (DHHC)-gold nanorod (GNR) conjugate was developed. DHHC was synthesized by successive dihydroxyphenylation and hydrazidation of hydroxyethyl chitosan through carbodiimide reaction and click chemistry, respectively. The conjugate was obtained by chemically bonding DHHC onto GNR via Au-catechol bonds. Doxorubicin (DOX) was loaded onto the conjugate via an acid-labile hydrazone linkage with a drug loading content of 5.1%. DOX-loaded conjugate displayed good stability in neutral aqueous solutions and exhibited pH-responsive drug release and surface charge reversal behaviors. In vitro biological studies indicated that the conjugate could be effectively internalized by breast cancer MCF-7 cells and synergistic therapeutic effects were demonstrated, suggesting its great potential in combined photothermal-chemotherapy of breast cancer.

A dual-targeted hyaluronic acid-gold nanorod platform with triple-stimuli responsiveness for photodynamic/photothermal therapy of breast cancer.

Multi-stimuli-responsive theranostic nanoplatform integrating functions of both imaging and multimodal therapeutics holds great promise for improving diagnosis and therapeutic efficacy. In this study, we reported a pH, glutathione (GSH) and hyaluronidase (HAase) triple-responsive nanoplatform for HER2 and CD44 dual-targeted and fluorescence imaging-guided PDT/PTT dual-therapy against HER2-overexpressed breast cancer. The nanoplatform was fabricated by functionalizing gold nanorods (GNRs) with hyaluronic acid (HA) bearing pendant hydrazide and thiol groups via Au-S bonds, and subsequently chemically conjugating 5-aminolevulinic acid (ALA), Cy7.5 and anti-HER2 antibody onto HA moiety for PDT, fluorescence imaging and active targeting, respectively. The resulting versatile nanoplatform GNR-HA-ALA/Cy7.5-HER2 had uniform sizes, favorable dispersibility, as well as pH, GSH and HAase triple-responsive drug release manner. In vitro studies demonstrated that HER2 and CD44 receptor-mediated dual-targeting strategy could significantly enhance the cellular uptake of GNR-HA-ALA/Cy7.5-HER2. Under near-infrared (NIR) irradiation, MCF-7 cells could efficiently generate reactive oxygen species (ROS) and heat, and be more efficiently killed by a combination of PDT and PTT as compared with individual therapy. Pharmacokinetic and biodistribution studies showed that the nanoplatform possessed a circulation half-life of 1.9 h and could be specifically delivered to tumor tissues with an accumulation ratio of 12.8%. Upon the fluorescence imaging-guided PDT/PTT treatments, the tumors were completely eliminated without obvious side effects. The results suggest that the GNR-HA-ALA/Cy7.5-HER2 holds great potential for breast cancer therapy. STATEMENT OF SIGNIFICANCE: A combination of photodynamic therapy (PDT) and photothermal therapy (PTT) is emerging as a promising cancer treatment strategy. However, its therapeutic efficacy is compromised by the nonspecific delivery and unintended release of photo-responsive agents. Herein, we developed a multifunctional theranostic nanoplatform GNR-HA-ALA/Cy7.5-HER2 with pH, glutathione and hyaluronidase triple-responsive drug release for HER2 and CD44 dual-targeted and fluorescence imaging-guided PDT/PTT therapy against breast cancer. We demonstrated that HER2 and CD44 receptors-mediated dual-targeting strategy significantly enhanced the cellular uptake of GNR-HA-ALA/Cy7.5-HER2. We also demonstrated that the combined PDT/PTT treatment had significantly superior antitumor effect than PDT or PTT alone both in vitro and in vivo. Therefore, GNR-HA-ALA/Cy7.5-HER2 could serve as a promising nanoplatform for HER2-positive breast cancer therapy.

PEGylated hydrazided gold nanorods for pH-triggered chemo/photodynamic/photothermal triple therapy of breast cancer.

Integration of multimodal therapies into one nanoplatform holds great promise to overcome the drawbacks of conventional single-modal therapy and pursues enhanced anticancer efficacy. Herein, we developed a PEGylated gold nanorods (GNRs)-based nanoplatform (GNRs-MPH-ALA/DOX-PEG) with pH-responsive drug release property for triple-combined chemotherapy (CT), photodynamic therapy (PDT) and photothermal therapy (PTT) of breast cancer. GNRs were first decorated with mercaptopropionylhydrazide (MPH) and thiol-terminated monomethoxyl poly(ethylene glycol) (mPEG-SH) via Au-thiol linkage, and subsequently conjugated with chemotherapeutant doxorubicin (DOX) and pro-photosensitizer 5-aminolevulinic acid (ALA) through acid-liable hydrazone bonds between drugs and MPH molecules. The resulting nanoplatform GNRs-MPH-ALA/DOX-PEG exhibited excellent stability in physiological solutions and pH-responsive DOX and ALA release behaviors. In vitro studies showed that GNRs-MPH-ALA/DOX-PEG could efficiently enter human breast cancer MCF-7 cells and release DOX and ALA into cytoplasm. Furthermore, DOX could locate in the cell nucleus and ALA was productively metabolized into protoporphyrin IX (PpIX). Upon near-infrared (NIR) irradiation, PpIX produced enough reactive oxygen species for PDT and meanwhile GNRs could efficiently induce hyperthermia for PTT. Compared with single CT and dual-modal CT/PDT or CT/PTT treatment, the triple-combined CT/PDT/PTT treatment could more efficiently kill MCF-7 cells via a superadditive antitumor effect. Furthermore, the circulation half-life of GNRs-MPH-ALA/DOX-PEG in the blood was as long as approximately 52 min and it exhibited a tumor accumulation of 3.3%. The triple-combined CT/PDT/PTT treatment could completely suppress tumor growth without obvious systemic toxicity. Our study paves a new avenue for multimodal therapy of breast cancer. STATEMENT OF SIGNIFICANCE: The development of a simple but effective strategy to construct a versatile nanoplatform for multi-combined therapy still remains an enormous challenge. In this work, we developed a novel and simple nanoplatform GNRs-MPH-ALA/DOX-PEG with pH-responsive drug release for triple-combined chemotherapy (CT), photodynamic therapy (PDT) and photothermal therapy (PTT) of breast cancer. The nanoplatform could be efficiently internalized by MCF-7 cells. The intracellular GNRs-MPH-ALA/DOX-PEG could release DOX for CT, induce hyperthermia for PTT and generate high levels of ROS for PTT. Compared with single CT and dual-modal CT/PDT or CT/PTT treatments, the triple-combined CT/PDT/PTT treatment could more efficiently kill MCF-7 cells via a superadditive antitumor effect. Furthermore, upon triple-combined CT/PDT/PTT treatment, the tumor growth was completely suppressed without obvious systemic toxicity.