Neferine alleviates ovariectomy-induced osteoporosis by enhancing osteogenic differentiation of bone marrow mesenchymal stem cells via regulation of the p38MAPK pathway.

OBJECTIVE: Osteoporosis, a skeletal ailment marked by bone metabolism imbalance and disruption of bone microarchitecture, Neferine, a bisbenzylisoquinoline alkaloid with diverse pharmacological activities, has received limited attention in the context of osteoporosis treatment. METHODS: We employed a bilateral ovariectomy (OVX) rat model to induce osteoporosis and subsequently administered Neferine treatment for four weeks following successful model establishment. Throughout the modeling and treatment phases, we closely monitored rat body weights. We assessed alterations in bone tissue microstructure through micro-CT, HE staining, and safranin O-fast green staining. Levels of bone formation and resorption markers in serum were evaluated using ELISA assay. Western blot analysis was employed to determine the expression levels of p38MAPK, p-p38MAPK, and bone formation-related genes in bone tissue. We isolated and cultured OVX rat BMSCs (OVX-BMSCs) and induced osteogenic differentiation while simultaneously introducing Neferine and the p38MAPK inhibitor SB203580 for intervention. RESULTS: Neferine treatment effectively curbed the rapid weight gain in OVX rats, ameliorated bone loss, and decreased serum levels of TRAP, CTX-I, PINP, and BALP. Most notably, Neferine promoted the expression of bone formation-related factors in bone tissue of OVX rats, while concurrently activating the p38MAPK signaling pathway. In in vitro experiments, Neferine facilitated the expression of bone formation-related factors in OVX-BMSCs, increased the osteogenic differentiation potential of OVX-BMSCs, and activated the p38MAPK signaling pathway. Nevertheless, SB203580 partially reversed Neferine's promotive effect. CONCLUSION: Neferine can boost the osteoblastic differentiation of BMSCs and alleviate OVX-induced osteoporosis in rats by activating the p38MAPK signaling pathway.

Neferine alleviates acute kidney injury by regulating the PPAR-α/NF-κB pathway.

Acute kidney injury (AKI) is a cluster of clinical syndromes with diverse etiologies that ultimately result in a swift decline in kidney function. Regrettably, AKI lacks effective therapeutic agents at present. Neferine, a bioactive alkaloid derived from Lotus Plumule, has been reported to alleviate AKI triggered by cisplatin, ischemia/reperfusion (I/R), and sepsis by inhibiting inflammatory pathways. However, the precise molecular mechanisms underpinning its renoprotective effects remain elusive. Peroxisome proliferator-activated receptor alpha (PPAR-α), a regulator of lipid metabolism with anti-inflammatory properties, was investigated in this study to examine its role in neferine's renoprotective effects in cellular and mouse models of AKI. We found that neferine pretreatment in both I/R- or lipopolysaccharide (LPS)-induced AKI models inhibited the activation of the NF-κB inflammatory pathway and reversed PPAR-α deficiency. In NRK-52E cells exposed to hypoxia/reoxygenation (H/R) or LPS, overexpression of PPAR-α resulted in inhibition of the NF-κB pathway and TNF-α production, while PPAR-α silencing via siRNA transfection negated neferine's anti-inflammatory effects. Furthermore, pretreatment with neferine not only reduced lipid accumulation but also reversed the downregulation of FAO-related enzymes induced by LPS. Our findings suggest that neferine's renoprotective effects against AKI are partially mediated through the reversal of renal PPAR-α deficiency and subsequent inhibition of the inflammatory NF-κB pathway. Therefore, regulating renal PPAR-α expression by neferine could represent a promising therapeutic strategy for AKI.

Construction and application of covalently bonded CD147 cell membrane chromatography model based on polystyrene microspheres.

In this study, a novel cell membrane chromatography (CMC) model was developed to investigate cluster of differentiation 147 (CD147) targeted anti-tumor drug leads for specific screening and ligand-receptor interaction analysis by SNAP-tagged CD147 fusion protein conjugation and polystyrene microspheres (PS) modification. Traditional Chinese medicines (TCMs) are widely used in the treatment of cancer. CD147 plays important roles in tumor progression and acts as an attractive target for therapeutic intervention; therapeutic drugs for CD147-related cancers are limited to date. Thus, a screening method for active components in TCMs is crucial for the further research and development of CD147 antagonists. However, improvement is still needed to perform specific and accurate drug lead screening using the CMC-based method. Recently, our group developed a covalently immobilized receptor-SNAP-tag/CMC model using silica gel as carrier. Besides the carboxyl group on multi-step modified silica particles, the amino group of benzyl-guanine (BG, substrate of SNAP-tag) also possesses reactivity towards the carboxyl group on available carboxyl-modified PS. Herein, we used PS as carrier and an extended SNAP-tag with CD147 receptor to construct the PS-BG-CD147/CMC model for active compound investigation coupled with HPLC/MS and applied this coupled PS-BG-CD147/CMC-HPLC/MS two-dimensional system to drug lead screening from Nelumbinis Plumula extract (NPE) sample. In addition, to comprehensively verify the pharmacological effects of screened ingredients, a cell proliferation inhibition assay was performed, and the interaction between the ingredients and CD147 was studied by the frontal analysis method. This study developed a high-throughput PS-based CMC screening platform, which could be widely applied and utilized in chromatographic separation and drug lead discovery.

Neferine protected cardiomyocytes against hypoxia/oxygenation injury through SIRT1/Nrf2/HO-1 signaling.

Acute myocardial infarction is regarded as myocardial necrosis resulting from myocardial ischemia/reperfusion (I/R) damage and retains a major cause of mortality. Neferine, which was extracted from the green embryos of mature seeds of Nelumbo nucifera Gaertn., has been reported to possess a broad range of biological activities. However, its underlying mechanism on the protective effect of I/R has not been fully clarified. A hypoxia/reoxygenation (H/R) model with H9c2 cells closely simulating myocardial I/R injury was used as a cellular model. This study intended to research the effects and mechanism underlying neferine on H9c2 cells in response to H/R stimulation. Cell Counting Kit-8 and lactate dehydrogenase (LDH) release assays were employed to measure cell viability and LDH, respectively. Apoptosis and reactive oxygen species (ROS) were determined by flow cytometry analysis. Oxidative stress was evaluated by detecting malondialdehyde, superoxide dismutase, and catalase. Mitochondrial function was assessed by mitochondrial membrane potential, ATP content, and mitochondrial ROS. Western blot analysis was performed to examine the expression of related proteins. The results showed that hypoxia/reoxygenation (H/R)-induced cell damage, all of which were distinctly reversed by neferine. Moreover, we observed that neferine inhibited oxidative stress and mitochondrial dysfunction induced by H/R in H9c2 that were concomitant with increased sirtuin-1 (SITR1), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 expression. On the contrary, silencing the SIRT1 gene with its small interferingRNA eliminated the beneficial effects of neferine. It is concluded that neferine preconditioning attenuated H/R-induced cardiac damage via suppressing apoptosis, oxidative stress, and mitochondrial dysfunction, which may be partially ascribed to the activation of SIRT1/Nrf2 signaling pathway.

Neferine mitigates cisplatin-induced acute kidney injury in mice by regulating autophagy and apoptosis.

PURPOSE: The nephrotoxicity caused by cisplatin severely limits the application and affects related platinum-based therapeutics. Neferine is a dibenzylisoquinoline alkaloid extracted from a Chinese medicinal herb (Nelumbo nucifera Gaertn), which can decrease cisplatin-induced apoptosis of NRK-52E cells by activating autophagy in vitro in our previous study. In this article, we aimed to further investigate the protective effect of neferine, against to the cispltain-induced kidney damage in mice. METHODS: Six groups were designed in our study. Renal index, mice serum creatinine and blood urea nitrogen levels were detected after the mice were killed. HE staining was used to observe the pathological changes of each group. The apoptosis of mouse kidney tissue was detected by TUNEL. Immunofluorescence and Western blot were used to detect the expression of cleaved-caspase3 and LC3. The transmission electron microscope was used to reveal the changes of apoptosis and autophagy of renal tubular epithelial cells in different groups. RESULTS: In our findings, the pathological changes of acute kidney injury were easily observed in cisplatin-treated mice while those in the neferine-pretreated groups were significantly alleviated. The apoptosis induced by cisplatin in mice increased evidently compared with the control group, which was decreased in the mice with neferine pretreatment. What' more, we found that autophagy increased obviously in mice pretreated by neferine contrast to the cisplatin-treated mice. CONCLUSION: In our study, neferine can effectively alleviate cisplatin-induced renal injury in mice, as well act as an autophagy-regulator in kidney protection.

Neferine alleviates ovalbumin-induced asthma via MAPK signaling pathways in mice.

PURPOSE: To investigate the role of neferine in ovalbumin (OVA)-induced asthma, and to reveal the possible mechanism. METHODS: In OVA-induced asthmatic mice, enzyme-linked-immunosorbent serologic assay was performed to evaluate the level of interleukin (IL)-4, IL-5, IL-13, immunoglobulin E (IgE) in serum and tumor necrosis factor-α (TNF-α), IL-6, IL-1ß, and monocyte chemoattractant protein-1 (MCP-1) in bronchoalveolar lavage fluid (BALF). Eosinophil, neutrophil, and lymphocyte counts in BALF were calculated to assess inflammation. The pulmonary function was measured by airway resistance, peak expiratory flow (PEF) and forced expiratory volume/forced vital capacity (FEV0.4/FVC) ratio, and respiratory rate. Hematoxylin and eosin staining and Masson staining were used to evaluate lung injury. Further, Western blot analysis was conducted to detect phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 of mitogen-activated protein kinase (MAPK) signaling pathways. RESULTS: Neferine, 20 mg/kg or 40 mg/kg, could significantly decrease the levels of IL-4, IL-5, IL-13, and IgE in OVA-induced serum, and that of TNF-α, IL-6, IL-1ß, and MCP-1 in OVA-induced BALF. Moreover, neferine could significantly decline eosinophil, neutrophil, and lymphocyte counts in BALF. Neferine contributed to improve OVA-induced airway resistance, promoted the value of PEF and FEV0.4/FVC ratio, and recovered the respiratory rate. It also reduced mucus secretion, distribution of inflammatory and goblet cells around bronchi, and attenuated collagen deposition in lung tissues. Furthermore, neferine reduced the phosphorylation of p38, JNK, and ERK to inhibit MAPK signaling pathways. CONCLUSION: Neferine relieves asthma-induced inflammatory reaction, airway resistance, and lung injury by inhibiting MAPK signaling pathways. This could serve neferine as a novel therapeutic candidate for treating asthma.

Neferine Targets the Oncogenic Characteristics of Androgen-Dependent Prostate Cancer Cells via Inducing Reactive Oxygen Species.

Castration resistance poses a significant challenge in the management of advanced prostate cancer (PCa), with androgen deprivation therapy (ADT) or chemotherapy being the primary treatment options. However, these approaches often lead to significant side effects and the development of therapeutic resistance. Therefore, it is crucial to explore novel treatment options that can efficiently target PCa, improve patient survival, and enhance their quality of life. Neferine (Nef), a bioactive compound derived from plants, has emerged as a promising candidate for cancer treatment due to its ability to induce apoptosis, autophagy, and cell cycle arrest. In this study, we investigated the potential anticancer effects of Nef in androgen receptor (AR)-positive LNCaP and VCaP cells, representative models of androgen-dependent PCa. Our findings demonstrate that Nef effectively inhibits cell growth, proliferation, and the tumorigenic potential of androgen-dependent PCa cells. Furthermore, Nef treatment resulted in the excessive production of reactive oxygen species (ROS), leading to the activation of key markers of autophagy and apoptosis. These results suggest that Nef has the potential to target the oncogenic characteristics of androgen-dependent PCa cells by exploiting the potency of ROS and inducing autophagy and apoptosis in AR-positive PCa cells. These findings shed light on the therapeutic potential of Nef as a novel treatment option with reduced side effects for androgen-dependent prostate cancer. Further investigations are warranted to assess its efficacy and safety in preclinical and clinical settings.

Optimization of Neferine Purification Based on Response Surface Methodology and Its Anti-Metastasis Mechanism on HepG2 Cells.

Liver cancer continues to be a focus of scientific research due to its low five-year survival rate. One of its main core issues is the high metastasis of cells, for which there is no effective treatment. Neferine was originally isolated from Plumula nelumbinis and demonstrated to have a good antitumor effect. In order to extract high-purity Neferine in a more efficient and environmentally friendly manner, response surface methodology (RSM) was used to optimize the isolation and purification procedures in this study. The extract conditions of a 7:3 ratio for the eluent of dichloromethane: methanol, 1:60 for the mass ratio of the extract amount: silica gel, and 3 mL/min of the elution flow rate were shown to be the optimal conditions. These conditions resulted in the highest yield of 6.13 mg per 66.60 mg of starting material, with productivity of 8.76% and purity of 87.04%. Compared with the previous methods, this method can prepare Neferine in large quantities more quickly. We subsequently evaluated the antitumor activity of the purified Neferine against HepG2 hepatic cancer cells. The purified Neferine was found to inhibit the proliferation of HepG2 cells through the CCK-8 assay, with an IC50 of 33.80 µM in 24 h, 29.47 µM in 48 h, 24.35 µM in 72 h and 2.78 µM in 96 h of treatment. Neferine at a concentration of 3 µM could significantly inhibit the migration and invasion abilities of the HepG2 cells in vitro. We also explored the mechanism of action of Neferine via Western blot. We showed that Neferine could reduce RhoA expression by effectively inhibiting the phosphorylation of MYPT1, thereby effectively exerting anti-metastasis activity against HepG2 cells. Thus, we have optimized the isolation procedures for highly pure Neferine by response surface methodology (RSM) in this study, and purified Neferine is shown to play an essential role in the anti-metastasis process of liver cancer cells. The Neferine purification procedure may make a wide contribution to the follow-up development of other anti-metastasis lead compounds.

Effects of neferine on retinal tissue in experimental diabetic rat model.

PURPOSE: To investigate vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) immunoreactivities, as well as apoptosis and oxidative stress levels in Streptozotocin (STZ)-induced diabetic rats, and determine how neferine affected these parameters. METHODS: Thirty-five male Sprague Dawley rats were divided into five groups of seven. Fasting blood glucose was measured 72 h after diabetes mellitus (DM) induction in 21 rats using 60 mg/kg STZ dissolved in 0.4 ml (0.1 M) sodium-citrate buffer (pH:4.5), with values > 250 mg/dl considered diabetic. Group 1 received no treatment. Group 3 (healthy rats) received daily intraperitoneal (IP) 4 mg/kg neferine. Following DM induction: Group 2 (sham) received daily IP 0.25 ml/kg 0.9% normal saline; Group 4 received single IP 0.01 mL (2.5 mg/kg) bevacizumab, followed by daily IP 0.25 mL/kg 0.9% normal saline; and Group 5 received daily IP 4 mg/kg neferine. Total antioxidant capacity (TAC) and total oxidative stress (TOS) levels in serum and ocular tissue homogenates were evaluated using ELISA. TUNEL method was used for determining apoptosis and immuno-histochemical staining for PCNA and VEGF immunoreactivities. RESULTS: Group 5 had significantly higher TAC and lower TOS in serum and ocular tissue homogenates than Group 4 (p < 0.05). Despite significantly lower VEGF levels and apoptosis (p < 0.05), there was no significant change in PCNA immunoreactivity in Group 5 (p > 0.05). CONCLUSIONS: DM was associated with lower TAC, higher TOS and apoptotic cells, as well as VEGF and PCNA immunoreactivities in the retina. Neferine altered parameters other than PCNA in the opposite direction, demonstrating reductive effects on DM.

Neferine attenuates development of testosterone-induced benign prostatic hyperplasia in mice by regulating androgen and TGF-ß/Smad signaling pathways.

Benign prostatic hyperplasia (BPH) is a common urinary disease among the elderly, characterized by abnormal prostatic cell proliferation. Neferine is a dibenzyl isoquinoline alkaloid extracted from Nelumbo nucifera and has antioxidant, anti-inflammatory and anti-prostate cancer effects. The beneficial therapeutic effects and mechanism of action of neferine in BPH remain unclear. A mouse model of BPH was generated by subcutaneous injection of 7.5 mg/kg testosterone propionate (TP) and 2 or 5 mg/kg neferine was given orally for 14 or 28 days. Pathological and morphological characteristics were evaluated. Prostate weight, prostate index (prostate/body weight ratio), expression of type Ⅱ 5α-reductase, androgen receptor (AR) and prostate specific antigen were all decreased in prostate tissue of BPH mice after administration of neferine. Neferine also downregulated the expression of pro-caspase-3, uncleaved PARP, TGF-ß1, TGF-ß receptor Ⅱ (TGFBR2), p-Smad2/3, N-cadherin and vimentin. Expression of E-cadherin, cleaved PARP and cleaved caspase-3 was increased by neferine treatment. 1-100 µM neferine with 1 µM testosterone or 10 nM TGF-ß1 were added to the culture medium of the normal human prostate stroma cell line, WPMY-1, for 24 h or 48 h. Neferine inhibited cell growth and production of reactive oxygen species (ROS) in testosterone-treated WPMY-1 cells and regulated the expression of androgen signaling pathway proteins and those related to epithelial-mesenchymal transition (EMT). Moreover, TGF-ß1, TGFBR2 and p-Smad2/3, N-cadherin and vimentin expression were increased but E-cadherin was decreased after 24 h TGF-ß1 treatment in WPMY-1 cells. Neferine reversed the effects of TGF-ß1 treatment in WPMY-1 cells. Neferine appeared to suppress prostate growth by regulating the EMT, AR and TGF-ß/Smad signaling pathways in the prostate and is suggested as a potential agent for BPH treatment.

Natural isoquinoline alkaloids: Pharmacological features and multi-target potential for complex diseases.

Complex diseases such as neurodegenerative disorders and cancer constitute a growing public health problem due to the rising incidence and lack in effective therapies. Since pharmacotherapy based on a single target has been insufficient for drug development in complex diseases, the emerging multi-target approach is a promising strategy for the search of new drug candidates. Plant-derived isoquinoline alkaloids comprise a vast source of multimodal agents with unique structural diversity, and variated range of pharmacological activities. This review offers an exhaustive compilation of the pharmacological relevance and multi-target potential of natural isoquinolines, emphasizing their features and promising activity in complex diseases such as Alzheimer, Parkinson, and Cancer. Selected examples were discussed in depth to illustrate the most relevant structural motifs and their possible relationship with the multimodal activity offering a comprehensive baseline in the search and optimization of isoquinoline scaffolds with polypharmacological potential for complex diseases.

Neferine attenuates doxorubicin-induced fibrosis and hypertrophy in H9c2 cells.

Doxorubicin (DOX), an anthracycline antineoplastic candidate is used to treat various malignancies. Around 41% of patients undergoing DOX treatment develop acute cardiotoxicity. Preventing DOX-induced cardiac fibrosis and hypertrophy helps in evading cardiac remodeling leading to cardiomyopathy and heart failure. Neferine, an alkaloid from the lotus has numerous pharmacological activities. The present study was designed to evaluate the protective effect of neferine on DOX-mediated fibrosis and hypertrophy. DOX-induced fibrosis involves activation of transforming growth factor-ß1 (TGF-ß1), matrix metalloproteinase 2 (MMP-2), and MMP-9 with concomitant downregulation of tissue inhibitors of MMPs (TIMP)-1 and TIMP-2 expressions in H9c2 cardiomyoblasts. Furthermore, DOX treatment also resulted in hypertrophy with the increased cell volume and overexpression of hypertrophy markers calcineurin, brain natriuretic peptide, and atrial natriuretic peptide. Finally, DOX treatment resulted in apoptosis through activation of p53. Pretreatment with neferine markedly activated SIRT1 expression and modulated the expression levels of TGF-ß1 and p53, thereby significantly reducing DOX-induced fibrosis, hypertrophy, and apoptosis in H9c2 cardiomyoblasts.

Reversal of cisplatin resistance by neferine/isoliensinine and their combinatorial regimens with cisplatin-induced apoptosis in cisplatin-resistant colon cancer stem cells (CSCs).

Cisplatin chemotherapy to the colorectal cancer cells (CRCs) is accompanied by dose-limiting adverse effects along with the acquisition of drug resistance implicating low therapeutic outcomes. The present study is aimed to evaluate the chemosensitizing efficacy of neferine/isoliensinine or combinatorial regimen of neferine/isoliensinine with cisplatin against CSCs (cisplatin resistant colon stem cells). CSCs were developed using pulse exposure of cisplatin to parental HCT-15 cells. Neferine/isoliensinine or combinatorial regimens of Neferine/isoliensinine and cisplatin exhibited a stronger cytotoxic activity against CSCs compared to control. IC50 doses were found to be 6.5 µM for neferine, 12.5 µM for isoliensinine, and 120 µM for cisplatin respectively. Furthermore, the combinatorial regimen of a low dose of cisplatin (40 µM) with 4 µM neferine/8 µM isoliensinine induced cell death in a synergistic manner as described by isobologram. Neferine/isoliensinine could confer extensive intracellular reactive oxygen species generation in CSCs. Neferine/isoliensinine or combinatorial regimens dissipated mitochondrial membrane potential and enhanced intracellular [Ca2+ ]i, which were measured by spectroflurimetry. Furthermore, these combinatorial regimens induced a significant increase in the sub G0 phase of cell cycle arrest and PI uptake and alleviated the expression of ERCC1 in CSCs. Combinatorial regimens or neferine/isoliensinine treatments downregulated the cell survival protein expression (PI3K/pAkt/mTOR) and activated mitochondria-mediated apoptosis by upregulating Bax, cytochrome c, caspase-3, and PARP cleavage expression while downregulating the BCl-2 expression in CSCs. Our study confirms the chemosensitizing efficacy of neferine/isoliensinine or combinatorial regimens of neferine/isoliensinine with a low dose of cisplatin against CSCs.

Neferine, an Alkaloid from Lotus Seed Embryos, Exerts Antiseizure and Neuroprotective Effects in a Kainic Acid-Induced Seizure Model in Rats.

Current anti-seizure drugs fail to control approximately 30% of epilepsies. Therefore, there is a need to develop more effective anti-seizure drugs, and medicinal plants provide an attractive source for new compounds. This study aimed to evaluate the possible anti-seizure and neuroprotective effects of neferine, an alkaloid from the lotus seed embryos of Nelumbo nucifera, in a kainic acid (KA)-induced seizure rat model and its underlying mechanisms. Rats were intraperitoneally (i.p.) administrated neferine (10 and 50 mg/kg) 30 min before KA injection (15 mg/kg, i.p.). Neferine pretreatment increased seizure latency and reduced seizure scores, prevented glutamate elevation and neuronal loss, and increased presynaptic protein synaptophysin and postsynaptic density protein 95 expression in the hippocampi of rats with KA. Neferine pretreatment also decreased glial cell activation and proinflammatory cytokine (interleukin-1ß, interleukin-6, tumor necrosis factor-α) expression in the hippocampi of rats with KA. In addition, NOD-like receptor 3 (NLRP3) inflammasome, caspase-1, and interleukin-18 expression levels were decreased in the hippocampi of seizure rats pretreated with neferine. These results indicated that neferine reduced seizure severity, exerted neuroprotective effects, and ameliorated neuroinflammation in the hippocampi of KA-treated rats, possibly by inhibiting NLRP3 inflammasome activation and decreasing inflammatory cytokine secretion. Our findings highlight the potential of neferine as a therapeutic option in the treatment of epilepsy.

Neferine suppresses autophagy-induced inflammation, oxidative stress and adipocyte differentiation in Graves' orbitopathy.

Previous studies in Graves' orbitopathy (GO) patient-derived fibroblasts showed that inhibition of autophagy suppresses adipogenic differentiation. Autophagy activation is associated with inflammation, production of reactive oxygen species and fibrosis. Neferine is an alkaloid extracted from Nelumbo nucifera, which induces Nrf2 expression and inhibits autophagy. Here, we have elucidated the role of neferine on interleukin (IL)-13-induced autophagy using patient-derived orbital fibroblasts as an in vitro model of GO. GO patient-derived orbital fibroblasts were isolated and cultured to generate an in vitro model of GO. Autophagy was determined by Western blot detection of the markers such as Beclin-1, Atg-5 and LC3 and by immunofluorescence detection of autophagosome formation. Analysis of differentiation towards an adipogenic lineage was performed by Oil red O staining. The expression of inflammatory factors was detected by ELISA and semiquantitative RT-PCR. Neferine inhibited autophagy in GO orbital fibroblasts, as indicated by the suppression of IL-13-induced autophagosome formation, overexpression of autophagy markers, increased LC3-II/LC3-I levels and finally down-regulation of p62. Neferine suppressed IL-13-induced inflammation, ROS generation, fibrosis and adipogenic differentiation in GO patient-derived orbital fibroblasts. The anti-inflammatory, antioxidant and antiadipogenic effects of neferine were accompanied by the up-regulation of Nrf2. These results indicated that orbital tissue remodelling and inflammation in GO may be mediated by autophagy, and neferine suppressed autophagy-related inflammation and adipogenesis through a mechanism involving Nrf2.

Inhibitory effect on SARS-CoV-2 infection of neferine by blocking Ca2+ -dependent membrane fusion.

The coronavirus disease 2019 (COVID-19) pandemic has focused attention on the need to develop effective therapeutics against the causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and also against other pathogenic coronaviruses. In this study, we report on a kind of bisbenzylisoquinoline alkaloid, neferine, as a pan-coronavirus entry inhibitor. Neferine effectively protected HEK293/hACE2 and HuH7 cell lines from infection by different coronaviruses pseudovirus particles (SARS-CoV-2, SARS-CoV-2 [D614G, N501Y/D614G, 501Y.V1, 501Y.V2, 501Y.V3 variants], SARS-CoV, MERS-CoV) in vitro, with median effect concentration (EC50 ) of 0.13-0.41 µM. Neferine blocked host calcium channels, thus inhibiting Ca2+ -dependent membrane fusion and suppressing virus entry. This study provides experimental data to support the fact that neferine may be a promising lead for pan-coronaviruses therapeutic drug development.

Pharmacokinetics, bioavailability and metabolism of neferine in rat by LC-MS/MS and LC-HRMS.

In this study, a simple and sensitive analytical method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for the determination of neferine in rat plasma. After acetonitrile-mediated protein precipitation, the samples were separated on an Acquity BEH C18 column (2.1 × 50 mm, 1.7 µm) maintained at 40°C. The mobile phase comprising 0.1% formic acid in water and acetonitrile was delivered at a flow rate of 0.4 ml/min. The mass detection was conducted using multiple reaction monitoring mode with ion transitions at 625.4 > 206.3 and m/z 622.9 > 380.9 for neferine and internal standard, respectively. The assay was demonstrated to be linear over the concentration range of 0.5-1,000 ng/ml, with correlation coefficient >0.999 (r > 0.999). The validated method was further applied to the pharmacokinetic study of neferine in rat plasma. In addition, the metabolism of neferine was investigated using high-resolution mass spectrometry. A total of six metabolites from rat liver microsomes and plasma were detected and their structures were identified according to their fragment ions. The proposed metabolic pathways of neferine were demethylation, dealkylation, dehydrogenation and glucuronidation.

Anti-Allergic and Anti-Inflammatory Effects of Neferine on RBL-2H3 Cells.

Mast cells play a very important role in skin allergy and inflammation, including atopic dermatitis and psoriasis. In the past, it was found that neferine has anti-inflammatory and anti-aging effects on the skin, but its effect on mast cells has not yet been studied in detail. In this study, we used mast cells (RBL-2H3 cells) and mouse models to study the anti-allergic and inflammatory effects of neferine. First, we found that neferine inhibits the degranulation of mast cells and the expression of cytokines. In addition, we observed that when mast cells were stimulated by A23187/phorbol 12-myristate-13-acetate (PMA), the elevation of intracellular calcium was inhibited by neferine. The phosphorylation of the MAPK/NF-κB pathway is also reduced by pretreatment of neferine. The results of in vivo studies show that neferine can improve the appearance of dermatitis and mast cell infiltration caused by dinitrochlorobenzene (DNCB). Moreover, the expressions of barrier proteins in the skin are also restored. Finally, it was found that neferine can reduce the scratching behavior caused by compound 48/80. Taken together, our results indicate that neferine is a very good anti-allergic and anti-inflammatory natural product. Its effect on mast cells contributes to its pharmacological mechanism.

Effect of Neferine on DNCB-Induced Atopic Dermatitis in HaCaT Cells and BALB/c Mice.

Atopic dermatitis (AD) is a chronic and persistent inflammatory skin disease characterized by eczematous lesions and itching, and it has become a serious health problem. However, the common clinical treatments provide limited relief and are accompanied by adverse effects. Therefore, there is a need to develop novel and effective therapies to treat AD. Neferine is a small molecule compound isolated from the green embryo of the mature seeds of lotus (Nelumbo nucifera). It has a bisbenzylisoquinoline alkaloid structure. Relevant studies have shown that neferine has many pharmacological and biological activities, including anti-inflammatory, anti-thrombotic, and anti-diabetic activities. However, there are very few studies on neferine in the skin, especially the related effects on inflammatory skin diseases. In this study, we proved that it has the potential to be used in the treatment of atopic dermatitis. Through in vitro studies, we found that neferine inhibited the expression of cytokines and chemokines in TNF-α/IFN-γ-stimulated human keratinocyte (HaCaT) cells, and it reduced the phosphorylation of MAPK and the NF-κB signaling pathway. Through in vivo experiments, we used 2,4-dinitrochlorobenzene (DNCB) to induce atopic dermatitis-like skin inflammation in a mouse model. Our results show that neferine significantly decreased the skin barrier damage, scratching responses, and epidermal hyperplasia induced by DNCB. It significantly decreased transepidermal water loss (TEWL), erythema, blood flow, and ear thickness and increased surface skin hydration. Moreover, it also inhibited the expression of cytokines and the activation of signaling pathways. These results indicate that neferine has good potential as an alternative medicine for the treatment of atopic dermatitis or other skin-related inflammatory diseases.

Neferine sensitized Taxol-resistant nasopharygeal carcinoma to Taxol by inhibiting EMT via downregulating miR-130b-5p.

Taxol resistance led to the poor survival prognosis in advanced nasopharyngeal carcinoma (NPC). Epithelial-mesenchymal transition (EMT) plays an important role in tumor chemoresistance. Neferine (NEF) is found to sensitize the cancer cells to chemotherapeutic agents, but its effects and mechanisms on NPC Taxol resistance is unclear. In this study, we discovered that Taxol-resistant cell lines 5-8F/Taxol and CNE-1/Taxol had the greater ability to metastasis and the higher expression of EMT markers. Then we found that NEF could inhibit the viability and EMT process in the Taxol-resistant cell lines. Furthermore, we confirmed that NEF could augment therapeutic efficacy of Taxol on NPC Taxol-resistant cell lines. Further through Microarray based analysis, we found that miR-130b-5p was stably down-regulated after treating 5-8F/Taxol with NEF. Later we verified that up-regulation of miR-130b-5p could not only promote the EMT-related migration/invasion, but also impair the inhibition effects of NEF on the EMT-associated metastatic ability and the chemotherapy resistance to Taxol. In conclusion, our results firstly suggested that NEF may enhanced Taxol sensitivity in NPC Taxol-resistant cell lines through inhibition of EMT which mediated by miR-130b-5p downregulation in vitro and in vivo. NEF may be used as a Taxol sensitizer in chemotherapy of NPC.