Synergistic antitumor effects of Peiminine and Doxorubicin on breast cancer through enhancing DNA damage via ZEB1.

Peiminine, the primary biologically active compound from Fritillaria thunbergii Miq., has demonstrated significant pharmacological activities. Doxorubicin is one of the most potent chemotherapeutic agents for breast cancer (BC). This study was designed to investigate the efficacy and underlying mechanisms of Peiminine combined with Doxorubicin in treating BC. Our results demonstrated that the combination of Peiminine and 1 mg/kg Doxorubicin exhibited more significant suppression of tumor growth compared with the monotherapy in MDA-MB-231 xenograft nude mice model, which is comparable to the effect of 3 mg/kg Doxorubicin in vivo. Notably, the 3 mg/kg Doxorubicin monotherapy resulted in organ toxicity, specifically in the liver and heart, whereas no toxicity was observed in the combination group. In vitro, this combined treatment exhibited a synergistic reduction on the viability of BC cells. Peiminine enhanced the cell cycle arrest and DNA damage induced by Doxorubicin. Furthermore, the combination treatment effectively blocked DNA repair by inhibiting the MAPKs signaling pathways. And ZEB1 knockdown attenuated the combined effect of Peiminine and Doxorubicin on cell viability and DNA damage. In conclusion, our study found that the combination of Peiminine and Doxorubicin showed synergistic inhibitory effects on BC both in vivo and in vitro through enhancing Doxorubicin-induced DNA damage. These findings support that their combination is a novel and promising therapeutic strategy for treating BC.

Peiminine triggers ferroptosis to inhibit breast cancer growth through triggering Nrf2 signaling.

BACKGROUND: Peiminine (PMI) is an active alkaloid sourced from Fritillaria thunbergii, which has been shown to suppress the development of a variety of tumors. Whereas, the roles and precise mechanism of PMI in breast cancer (BC) development remain not been clarified. METHODS: The cytotoxic effect of PMI on MCF-10A and BC cell lines (MCF-7 and BT-549) were assessed by MTT and LDH release assay. Cell proliferation was evaluated by EdU staining. Levels of Malondialdehyde (MDA), reactive oxygen species (ROS), glutathione (GSH) activity and iron assay were measured by Enzyme linked immunosorbent assay (ELISA) kits, respectively. Transmission Electron Microscope was performed to observe mitochondrial morphological structure. Immunofluorescence, immunohistochemistry, and western blot were conducted to examine protein levels, respectively. Xenograft model was used to confirm cellular findings. RESULTS: PMI treatment reduced the viability and enhanced LDH level of MCF-7 and BT-549 cells in a time- and concentration-dependent manner, and further suppressed cell proliferation in vitro and tumor growth in vivo. Subsequently, PMI administration resulted in significant increases of ROS, MDA and iron levels, reduction of GSH activity as well as mitochondrial shrinkage and GPX4 reduction, while all these phenomena could be rescued by ferrostatin-1. Mechanistically, PMI treatment led to promoted Nrf2 expression and its nuclear translocation, as well as it's downstream protein HO-1 and NQO1 expressions. Notably, ML-385, a Nrf2 specific inhibitor, greatly reversed the anti-tumor effects and pro-ferroptosis role of PMI in BC cells. CONCLUSION: Taking these finding together, PMI could stimulate ferroptosis to inhibit BC tumor growth by activating Nrf2-HO-1 signaling pathway.

Anti-inflammatory activity of peiminine in acetic acid-induced ulcerative colitis model.

Ulcerative colitis is a chronic inflammatory disorder of the intestinal mucosa and a prevalent gastrointestinal condition in developed countries. Peiminine, derived from the Fritillaria imperialis plant, exhibits remarkable anti-inflammatory and anti-cancer properties. This study aims to investigate the anti-inflammatory effects of peiminine in an experimental model of ulcerative colitis. Ulcerative colitis was induced intra-rectally in all groups, except the negative control, using 100 µl of 4% acetic acid. Peiminine treatment was initiated after ulcerative colitis induction and symptom manifestation. After the final injection, mice were sacrificed on day 15 for assessment. Various parameters were evaluated, including disease activity index, myeloperoxidase activity, nitric oxide levels, production and expression of IL-1, IL-6, TNF-α cytokines, and expression of IL-1ß, IL-6, TNF-α, iNOS, and COX2 genes. Microscopic pathological evaluation was performed on colon tissue. Peiminine treatment resulted in reduced levels of NO, MPO, IL-1ß, IL-6, and TNF-α. Furthermore, the expression of IL-1ß, IL-6, TNF-α genes, iNOS, and COX2 genes was decreased in response to peiminine treatment in these mice. This study demonstrates the effectiveness of peiminine in alleviating inflammatory manifestations and mitigating intestinal tissue damage in an experimental model of ulcerative colitis, probably by anti-inflammatory procedure. Peiminine holds potential as a therapeutic adjunct for the management of ulcerative colitis.

Interaction of a Novel Alternatively Spliced Variant of HSD11B1L with Parkin Enhances the Carcinogenesis Potential of Glioblastoma: Peiminine Interferes with This Interaction.

Glioblastoma (GBM) is a primary brain tumor of unknown etiology. It is extremely aggressive, incurable and has a short average survival time for patients. Therefore, understanding the precise molecular mechanisms of this diseases is essential to establish effective treatments. In this study, we cloned and sequenced a splice variant of the hydroxysteroid 11-ß dehydrogenase 1 like gene (HSD11B1L) and named it HSD11B1L-181. HSD11 B1L-181 was specifically expressed only in GBM cells. Overexpression of this variant can significantly promote the proliferation, migration and invasion of GBM cells. Knockdown of HSD11B1L-181 expression inhibited the oncogenic potential of GBM cells. Furthermore, we identified the direct interaction of parkin with HSD11B1L-181 by screening the GBM cDNA expression library via yeast two-hybrid. Parkin is an RBR E3 ubiquitin ligase whose mutations are associated with tumorigenesis. Small interfering RNA treatment of parkin enhanced the proliferative, migratory and invasive abilities of GBM. Finally, we found that the alkaloid peiminine from the bulbs of Fritillaria thunbergii Miq blocks the interaction between HSD11B1L-181 and parkin, thereby lessening carcinogenesis of GBM. We further confirmed the potential of peiminine to prevent GBM in cellular, ectopic and orthotopic xenograft mouse models. Taken together, these findings not only provide insight into GBM, but also present an opportunity for future GBM treatment.

Peimine ameliorates pulmonary fibrosis via the inhibition of M2-type macrophage polarization through the suppression of P38/Akt/STAT6 signals.

Peimine, a bioactive substance isolated from Chinese medicine Fritillaria, can potentially suppress pulmonary fibrosis (PF); however, its therapeutic mechanism remains unclear. Recent evidence suggests the participation of M2-type macrophages in the pathogenesis of PF. The present study aimed to investigate the effect of peimine on a bleomycin (BLM)-induced PF rat model and the underlying mechanism of this effect. After BLM administration, peimine was administered to rats from day 29 to day 42, with pirfenidone (PFD) as a positive control. H&E and Masson's trichrome stain were used to analyze histological changes. Q-PCR and western blotting were used to measure mRNA levels and protein levels, respectively. High-throughput RNA sequencing (RNA-seq) technology detected the differentially expressed genes (DEGs) regulated by peimine. Our results revealed that peimine treatment significantly ameliorated BLM-induced PF by suppressing histological changes and collagen deposition. In addition, peimine decreased the number of M2 macrophages and the expression of profibrotic factors. RNA-seq results showed that DEGs regulated by peimine in IL-4-induced macrophages were mainly associated with immune system processes, the PI3K/Akt pathway, and the MAPKs pathway. Then, immunofluorescence assay and western blot results demonstrated that peimine treatment suppressed the expression of p-p38 MAPK and p-Akt (s473) and also inhibited the nuclear translocation of p-STAT6. In conclusion, the present study demonstrated that peimine has a protective effect on PF through the suppression of M2 polarization of macrophages by inhibiting the STAT6, p38 MAPK, and Akt signals.

Peimine suppresses collagen-induced arthritis, activated fibroblast-like synoviocytes and TNFα-induced MAPK pathways.

BACKGROUND AND PURPOSE: Peimine (PM), a main isosterol alkaloid component isolated from the bulbs of traditional Chinese herb Fritillaria cirrhosa D. Don, has been demonstrated to exhibit multiple pharmacological properties, including anti-inflammation, anti-cancer and pain suppression. However, its effect on rheumatoid arthritis (RA) remains unknown. In the present study, we investigated the effect of PM on collagen-induced arthritis (CIA) rats in vivo and its inhibition on destructive behaviors of arthritic fibroblast-like synoviocytes (FLSs) in vitro. METHODS: Arthritis was induced in rats by chicken type II collagen. Arthritis score, radiological evaluation, and histopathological assessment were used to evaluate the therapeutic effects of PM on CIA rats. EdU assay, wound healing assay and real-time PCR were used to examine the inhibitory effect of PM on proliferation, migration, and over-expression of pro-inflammatory cytokines in TNFα-induced arthritic FLSs. TRAP staining and scanning electron microscopy were used to analyze the effect of PM on osteoclastogensis and bone resorption. Western blot was used to reveal PM's molecular mechanism of action on RA. RESULTS: PM significantly suppressed synovitis and bone destruction in CIA rats. In vitro experiments showed that PM treatment significantly inhibited TNFα-induced destructive behaviors of arthritic FLSs, including over-proliferation, migration and over-expression of pro-inflammatory cytokines. Additionally, RANKL-induced osteoclast formation and bone-resorpting function were also inhibited by PM. Further molecular mechanism studies revealed that PM treatment significantly suppressed TNFα-induced activations of MAPKs (ERK, JNK and p38) in arthritic FLSs. CONCLUSION: Our findings provide strong evidence that PM has the potential to be developed as a therapeutic agent for patients with RA.

Sipeimine ameliorates PM2.5-induced lung injury by inhibiting ferroptosis via the PI3K/Akt/Nrf2 pathway: A network pharmacology approach.

Fine particulate matter (PM2.5) exposure can cause lung injury and a large number of respiratory diseases. Sipeimine is a steroidal alkaloid isolated from Fritillaria roylei which has been associated with anti-inflammatory, antitussive and antiasthmatic properties. In this study, we explored the potential effects of sipeimine against PM2.5-induced lung injury in Sprague Dawley rats. Sipeimine alleviated lung injury caused by PM2.5 and decreased pulmonary edema, inflammation and the levels of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in the bronchoalveolar lavage fluid. In addition, sipeimine upregulated the glutathione (GSH) expression and downregulated the expression of 4-hydroxynonenal (4-HNE), tissue iron and malondialdehyde (MDA). The downregulation of proteins involved in ferroptosis, including nuclear factor E2-related factor 2 (Nrf2), glutathione peroxidase 4 (GPX4), heme oxygenase-1 (HO-1) and solute carrier family 7 member 11 (SLC7A11) was reversed by sipeimine. The administration of RSL3, a potent ferroptosis-triggering agent, blocked the effects of sipeimine. Using network pharmacology, we found that the effects of sipeimine were presumably mediated through the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. A PI3K inhibitor (LY294002) blocked the PI3K/Akt signaling pathway and reversed the effects of sipeimine. Overall, this study suggested that the protective effect of sipeimine against PM2.5-induced lung injury was mainly mediated through the PI3K/Akt pathway, ultimately leading to a reduction in ferroptosis.

Synergistic anti-inflammatory effects of peimine, peiminine, and forsythoside a combination on LPS-induced acute lung injury by inhibition of the IL-17-NF-κB/MAPK pathway activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Forsythia suspensa (Thunb.) Vahl and Fritillaria thunbergii Miq are traditional Chinese medicines that exhibit the ability to clear heat and toxic material effects. In China, the combination of these two medicines is widely used to treat mucopurulent sputum and bloody phlegm, arising due to phlegm-heat obstruction in respiratory diseases. However, very limited information is available regarding the combined anti-inflammatory effect of important effective components of Forsythia suspensa (Thunb.) Vahl and Fritillaria thunbergii Miq, namely peimine, peiminine, and forsythoside A. AIM OF THIS STUDY: To investigate synergistic anti-inflammatory effects of combined administration of peimine, peiminine, and forsythoside A on LPS-induced acute lung injury compared to combined administration of two compounds or individual administration, and unravel the underlying mechanism. MATERIAL AND METHODS: In the present study, male BALB/c mice received an oral dosage of sodium carboxymethylcellulose (CMC-Na) (0.5%, 1 mL/100 g), peimine, peiminine, forsythoside A, peimine + forsythoside A, peiminine + forsythoside A, and peimine + peiminine + forsythoside A (suspended in CMC-Na; 0.5%), once daily for 7 days. Subsequently, intratracheal instillation of LPS was applied to establish acute lung injury model. After 6 h of administration, the mice were sacrificed, and bronchoalveolar lavage fluid (BALF) and lung tissues were collected. These samples were further used to determine lung W/D (wet/dry) weight ratio, total protein (TP) levels, inflammatory cytokines (IL-6, TNF-α, IL-1ß, and IL-17), and expression of proteins involved in TLR4/MAPK/NF-κB pathway and IL-17 pathway. Further, tissue sections were subjected to H&E staining to assess the pathological alterations induced by LPS. The expression of IL-6 and TNF-α proteins in lung tissues was also analyzed using immunohistochemical staining. RESULTS: A synergistic anti-inflammatory effect of peimine, peiminine, and forsythoside A was observed when administered in combination to LPS-induced acute lung injury. The combined administration of peimine, peiminine, and forsythoside A had a strongly inhibitory effects on the W/D weight ratio, total protein (TP) level and the inflammatory cytokines (TNF-α, IL-6, IL-1ß, and IL-17) level in acute lung injury mice, compared to combined administration of two compounds or individual administration. The infiltration of inflammatory cells and thickened bronchoalveolar walls induced by LPS were also ameliorated through the combined administration of peimine, peiminine, and forsythoside A. More importantly, the upregulation of protein related to TLR4/MAPK/NF-κB signaling pathway and the activation of IL-17 were significantly suppressed by pretreatment with each of the three compounds alone, while the effects of individual compounds were synergistically augmented by the combined pretreatment of these three compounds. CONCLUSION: The combined administration of peimine, peiminine, and forsythoside A ameliorated inflammatory response in acute lung injury mice induced by LPS in a synergistic manner, the mechanism may be related to the dampening of the TLR4/MAPK/NF-κB signaling pathway and IL-17 activation.

Peiminine Suppresses RANKL-Induced Osteoclastogenesis by Inhibiting the NFATc1, ERK, and NF-κB Signaling Pathways.

Osteoclasts (OCs) play an important role in osteoporosis, a disease that is mainly characterized by bone loss. In our research, we aimed to identify novel approach for regulating osteoclastogenesis and thereby treating osteoporosis. Previous studies have set a precedent for screening traditional Chinese herbal extracts for effective inhibitors. Peiminine is an alkaloid extracted from the bulb of Fritillaria thunbergii Miq that reportedly has anticancer and anti-inflammatory effects. Thus, the potential inhibitory effect of peiminine on OC differentiation was investigated via a series of experiments. According to the results, peiminine downregulated the levels of specific genes and proteins in vitro and consequently suppressed OC differentiation and function. Based on these findings, we further investigated the underlying molecular mechanisms and identified the NF-κB and ERK1/2 signaling pathways as potential targets of peiminine. In vivo, peiminine alleviated bone loss in an ovariectomized mouse model.

Peiminine inhibits the progression of colorectal cancer through up-regulating miR-760 via declining the expression of long noncoding RNA LINC00659.

Colorectal cancer (CRC) is a commonly diagnosed type of cancer globally. The molecular mechanism by which peiminine suppressed the progression of CRC is not fully addressed. The viability was assessed through cell counting kit 8 assay. Colony formation assay was used to analyze the colony formation ability. The metastasis was evaluated by transwell migration and invasion assays. Quantitative real-time PCR was performed to measure the expression of LINC00659 and miR-760 in CRC cells. The binding sites between miR-760 and LINC00659 were predicted by Starbase software and verified by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA-pull down assay. The in-vivo function of peiminine in CRC progression was confirmed by murine xenograft model. Peiminine inhibited the viability, colony formation and metastasis of CRC cells. Peiminine notably down-regulated the expression of LINC00659, while the expression of miR-760 was up-regulated by peiminine treatment. MiR-760 was a direct target of LINC00659 in CRC cells. The depletion of miR-760 attenuated the inhibitory effects of LINC00659 intervention on the viability, colony formation and metastasis of CRC cells. Peiminine restrained the progression of CRC through LINC00659 and miR-760. LINC00659 inhibited the growth of CRC tumors through LINC00659/miR-760 axis in vivo. Peiminine suppressed the development of CRC through inhibiting the viability, colony formation and metastasis of CRC cells via LINC00659/miR-760 axis. LINC00659/miR-760 axis might be an underlying target for CRC therapy.

Peimine inhibits the growth and motility of prostate cancer cells and induces apoptosis by disruption of intracellular calcium homeostasis through Ca2+ /CaMKII/JNK pathway.

Prostate cancer (PC) is one of the most common malignant tumors in man. Peimine (PM) is a bioactive substance isolated from Fritillaria. Previous studies have shown that PM could inhibit the occurrence of a variety of cancers. However, the roles of PM in PC and its related mechanism have not been elucidated. Calcium (Ca2+ ) is an important intracellular messenger involved in a variety of cell processes. In this study, we found that the appropriate doses of PM (2.5, 5, and 10 µM) significantly inhibited the growth of PC cells (DU-145, LNCap, and PC-3), but has no significant effect on normal prostate cells (RWPE-1). In addition, PM treatment inhibited the invasion and migration of PC-3 cells and blocked the epithelial-mesenchymal transition process. These effects were exhibited a dose-dependent manner. Furthermore, the current results also showed that PM treatment significantly increased the Ca2+ concentration, the increased Ca2+ promoted the phosphorylation of Ca2+ /calmodulin-dependent protein kinase II (CaMKII) and c-Jun N-terminal kinase (JNK), further inhibited the growth and invasion of PC-3 cells, and induced its apoptosis. Ca2+ chelator BAPTA-AM (1 µM) could counteract the increase of intracellular Ca2+ concentration. Similarly, JNK pathway inhibitor SP600125 (10 µM) also inhibited cell growth and invasion and induced apoptosis. In addition, experiments in nude mice showed that PM inhibited tumor formation through Ca2+ /CaMKII/JNK signaling pathway. In conclusion, our results show that PM inhibits the growth and motility of prostate cancer cells and induces apoptosis by disruption of intracellular calcium homeostasis through Ca2+ /CaMKII/JNK pathway.

Isosteroid alkaloids with different chemical structures from Fritillariae cirrhosae bulbus alleviate LPS-induced inflammatory response in RAW 264.7 cells by MAPK signaling pathway.

Isosteroid alkaloids, natural products from Fritillariae Cirrhosae Bulbus, are well known for its antitussive, expectorant, anti-asthmatic and anti-inflammatory properties. However, the anti-inflammatory effect and its mechanism have not been fully explored. In this study, the anti-inflammatory activitives and the potential mechanisms of five isosteroid alkaloids from F. Cirrhosae Bulbus were investigated in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells. The pro-inflammatory mediators and cytokines were measured by Griess reagent, ELISA and qRT-PCR. The expression of MAPKs was investigated by western blotting. Treatment with the five isosteroid alkaloids in appropriate concentrations could reduce the production of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in supernatant, and suppressed the mRNA expressions of TNF-α and IL-6. Meanwhile, the five isosteroid alkaloids significantly inhibited the phosphorylated activation of mitogen activated protein kinase (MAPK) signaling pathways, including extracellular signal-regulated kinase (ERK1/2), p38 MAPK and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). These results demonstrated that isosteroid alkaloids from F. Cirrhosae Bulbus exert anti-inflammatory effects by down-regulating the level of inflammatory mediators via mediation of MAPK phosphorylation in LPS-induced RAW264.7 macrophages, thus could be candidates for the prevention and treatment of inflammatory diseases.

Mechanistic and therapeutic study of novel anti-tumor function of natural compound imperialine for treating non-small cell lung cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Bulbus Fritillaria cirrhosa D. Don (BFC) is a Chinese traditional herbal medicine that has long been used as an indispensable component in herbal prescriptions for bronchopulmonary diseases due to its well-established strong anti-inflammation and pulmonary harmonizing effects. Interestingly, there are few case reports in traditional Chinese medicine available where they found it to contribute in anti-tumor therapies. Imperialine is one of the most favored active substances extracted from BFC and has been widely recognized as an anti-inflammatory agent. AIM OF THE STUDY: The aim of the current work is to provide first-hand evidences both in vitro and in vivo showing that imperialine exerts anti-cancer effects against non-small cell lung cancer (NSCLC), and to explore the molecular mechanism of this anti-tumor activity. It is also necessary to examine its systemic toxicity, and to investigate how to develop strategies for feasible clinical translation of imperialine. MATERIALS AND METHODS: To investigate anti-NSCLC efficacy of imperialine using both in vitro and in vivo methods where A549 cell line were chosen as in vitro model NSCLC cells and A549 tumor-bearing mouse model was constructed for in vivo study. The detailed underlying anti-cancer mechanism has been systematically explored for the first time through a comprehensive set of molecular biology methods mainly including immunohistochemistry, western blot and enzyme-linked immunosorbent assays. The toxicity profile of imperialine treatments were evaluated using healthy nude mice by examining hemogram and histopathology. An imperialine-loaded liposomal drug delivery system was developed using thin film hydration method to evaluate target specific delivery. RESULTS: The results showed that imperialine could suppress both NSCLC tumor and associated inflammation through an inflammation-cancer feedback loop in which NF-κB activity was dramatically inhibited by imperialine. The NSCLC-targeting liposomal system was successfully developed for targeted drug delivery. The developed platform could favorably enhance imperialine cellular uptake and in vivo accumulation at tumor sites, thus improving overall anti-tumor effect. The toxicity assays revealed imperialine treatments did not significantly disturb blood cell counts in mice or exert any significant damage to the main organs. CONCLUSIONS: Imperialine exerts anti-cancer effects against NSCLC both in vitro and in vivo, and this previously unknown function is related to NF-κB centered inflammation-cancer feedback loop. Imperialine mediated anti-cancer activity is not through cytotoxicity and exhibit robust systemic safety. Furthermore, the liposome-based system we commenced would dramatically enhance therapeutic effects of imperialine while exhibiting extremely low side effects both on cellular and in NSCLC model. This work has identified imperialine as a promising novel anti-cancer compound and offered an efficient target-delivery solution that greatly facilitate practical use of imperialine.

Exosome-like nanoplatform modified with targeting ligand improves anti-cancer and anti-inflammation effects of imperialine.

Currently, most anti-cancer therapies are still haunted by serious and deleterious adverse effects. Here, we report a highly biocompatible tumor cell-targeting delivery systems utilizing exosome-like vesicles (ELVs) that delivers a low-toxicity anti-cancer agent imperialine against non-small cell lung cancer (NSCLC). First, we introduced a novel micelle-aided method to efficiently load imperialine into intact ELVs. Then, integrin α3ß1-binding octapeptide cNGQGEQc was modified onto ELV platform for tumor targeting as integrin α3ß1 is overexpressed on NSCLC cells. This system not only significantly improved imperialine tumor accumulation and retention, but also had extremely low systemic toxicity both in vitro and in vivo. Our discoveries offer new ways to utilize ELV more efficiently for both drug loading and targeting. The solid pharmacokinetics improvement and extraordinary safety of this system also highlight possibilities of alternative long course cancer therapies using similar strategies.

Peiminine inhibits the IL-1ß induced inflammatory response in mouse articular chondrocytes and ameliorates murine osteoarthritis.

Osteoarthritis (OA) is a common arthrosis characterized by degeneration and inflammation of articular cartilage. In recent decades, peiminine (Pm) has been identified as one of the active ingredients of Fritillaria plants. According to reports, Pm has a potent anti-inflammatory effect in various diseases. However, the effectiveness of Pm as an anti-inflammatory in OA has not previously been reported. This research aims to evaluate the anti-inflammatory effect of Pm on interleukin (IL)-1ß-induced mice chondrocytes and its chondroprotective effect in a mouse OA model with surgical destabilization of the medial meniscus. IL-1ß-induced expression of nitric oxide (NO), prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS) and interleukin-6 (IL-6) were all inhibited significantly by Pm pretreatment in vitro. In addition, Pm also inhibited the expression of thrombospondin motifs 5 (ADAMTS-5) and matrix metalloproteinase-13 (MMP-13), which are responsible for the degradation of the extracellular matrix (ECM). Additionally, the degradation of aggrecan and collagen II was reversed by Pm. Furthermore, Pm inhibited Akt phosphorylation and the nuclear transfer of nuclear factor-κB (NF-κB) and activated Nrf2/HO-1 signaling pathways both in vitro and in vivo. These findings suggested that Pm alleviated inflammatory effects in the IL-1ß-induced chondrocytes. Therefore, Pm might be a potential therapeutic agent for OA.

Application of UPLC-MS/MS to simultaneously detect four bioactive compounds in the tumour-shrinking decoction (FM1523) for uterine fibroids treatment.

INTRODUCTION: The Chinese medicine formulation, tumour-shrinking decoction (TSD, FM1523), which consists of 15 natural medicines, is used for uterine fibroids (UFs) therapy and possesses excellent clinical therapeutic effect. OBJECTIVE: To develop a sensitive and validated analytical method for the simultaneous quantification of four crucial bioactive compounds including isorhamnetin-3-O-neohesperidoside, curcumin, peimine and tetrahydropalmatine in the principal formulation of this decoction. METHODS: An ultra-performance liquid chromatography coupled tandem mass spectrometry (UPLC-MS/MS) with an electrospray ionisation (ESI) source in multiple reaction monitoring (MRM) mode was conducted to investigate these bioactive compounds in the TSD. The chromatographic separation was performed on a C18 column when the flow rate was adjusted at 0.2 mL/min with gradient elution of acetonitrile-water with 0.1% formic acid. Accelerated solvent extraction (ASE) method with higher extraction efficiency was employed for TSD sample pre-treatment. RESULTS: The linearity, limit of detection (LOD) and limit of quantification (LOQ) were determined for this analytical method. The mean recoveries of the compounds were determined between 100.23% and 104.02% with satisfactory relative standard deviation (RSD) in the ranges of 2.65% to 3.81%. The precision was evaluated by intra-day and inter-day tests, which revealed RSD within the ranges of 1.21% to 2.14% and 1.24% to 2.32%, respectively. CONCLUSION: The bioactive compounds of TSD samples were successfully quantified via UPLC-MS/MS with MRM mode. This study could help to evaluate the pharmacokinetic study of TSD during clinical applications and present a facile strategy for quantifying bioactive compounds in traditional Chinese Medicine decoction.

The effects and mechanism of peiminine-induced apoptosis in human hepatocellular carcinoma HepG2 cells.

Peiminine is a compound isolated from Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae family), which has demonstrated antitumor activities. But its precise molecular mechanism underlying antitumor activity remain elusive. In this study, peiminine-induced apoptosis towards human hepatocellular carcinoma and its molecular mechanism were investigated. MTT assay was employed to assess anticancer effects of peiminine upon Hela, HepG2, SW480 and MCF-7 cell lines. Nuclear staining and flow cytometry were carried out to detect apoptosis induced by peiminine. Mitochondrial membrane potential evaluation and Western blot analysis were performed to investigate the mechanism of peiminine-induced apoptosis. The results showed peiminine reduced the viability of HepG2 cells in a time- and dose-dependent manner and had an IC50 of 4.58 µg/mL at 24h. Peiminine significantly increased the percentage of apoptotic cells and the mitochondrial membrane potential dose-dependently in HepG2 cells. The results of Western blotting indicated the expressions of Bcl-2, procaspase-3, procaspase-8, procaspase-9, and PARP decreased in HepG2 cells treated with peiminine, while the expressions of Bax, caspase-3, caspase-8, caspase-9, and cleaved PARP1 increased. The result suggests that peiminine can induce apoptosis in human hepatocellular carcinoma HepG2 cells through both extrinsic and intrinsic apoptotic pathways.

Peiminine Inhibits Glioblastoma in Vitro and in Vivo Through Cell Cycle Arrest and Autophagic Flux Blocking.

BACKGROUND/AIMS: Glioblastoma multiforme (GBM) is the most devastating and widespread primary central nervous system tumour in adults, with poor survival rate and high mortality rates. Existing treatments do not provide substantial benefits to patients; therefore, novel treatment strategies are required. Peiminine, a natural bioactive compound extracted from the traditional Chinese medicine Fritillaria thunbergii, has many pharmacological effects, especially anticancer activities. However, its anticancer effects on GBM and the underlying mechanism have not been demonstrated. This study was conducted to investigate the potential antitumour effects of peiminine in human GBM cells and to explore the related molecular signalling mechanisms in vitro and in vivo Methods: Cell viability and proliferation were detected with MTT and colony formation assays. Morphological changes associated with autophagy were assessed by transmission electron microscopy (TEM). The cell cycle rate was measured by flow cytometry. To detect changes in related genes and signalling pathways in vitro and in vivo, RNA-seq, Western blotting and immunohistochemical analyses were employed. RESULTS: Peiminine significantly inhibited the proliferation and colony formation of GBM cells and resulted in changes in many tumour-related genes and transcriptional products. The potential anti-GBM role of peiminine might involve cell cycle arrest and autophagic flux blocking via changes in expression of the cyclin D1/CDK network, p62 and LC3. Changes in Changes in flow cytometry results and TEM findings were also observed. Molecular alterations included downregulation of the expression of not only phospho-Akt and phospho-GSK3ß but also phospho-AMPK and phospho-ULK1. Furthermore, overexpression of AKT and inhibition of AKT reversed and augmented peiminine-induced cell cycle arrest in GBM cells, respectively. The cellular activation of AMPK reversed the changes in the levels of protein markers of autophagic flux. These results demonstrated that peiminine mediates cell cycle arrest by suppressing AktGSk3ß signalling and blocks autophagic flux by depressing AMPK-ULK1 signalling in GBM cells. Finally, peiminine inhibited the growth of U251 gliomas in vivo. CONCLUSION: Peiminine inhibits glioblastoma in vitro and in vivo via arresting the cell cycle and blocking autophagic flux, suggesting new avenues for GBM therapy.

Peiminine Protects against Lipopolysaccharide-Induced Mastitis by Inhibiting the AKT/NF-κB, ERK1/2 and p38 Signaling Pathways.

Peiminine, an alkaloid extracted from Fritillaria plants, has been reported to have potent anti-inflammatory properties. However, the anti-inflammatory effect of peiminine on a mouse lipopolysaccharide (LPS)-induced mastitis model remains to be elucidated. The purpose of this experiment was to investigate the effect of peiminine on LPS-induced mastitis in mice. LPS was injected through the canals of the mammary gland to generate the mouse LPS-induced mastitis model. Peiminine was administered intraperitoneally 1 h before and 12 h after the LPS injection. In vitro, mouse mammary epithelial cells (mMECs) were pretreated with different concentrations of peiminine for 1 h and were then stimulated with LPS. The mechanism of peiminine on mastitis was studied by hematoxylin-eosin staining (H&E) staining, western blotting, and enzyme-linked immunosorbent assay (ELISA). The results showed that peiminine significantly decreased the histopathological impairment of the mammary gland in vivo and reduced the production of pro-inflammatory mediators in vivo and in vitro. Furthermore, peiminine inhibited the phosphorylation of the protein kinase B (AKT)/ nuclear factor-κB (NF-κB), extracellular regulated protein kinase (ERK1/2), and p38 signaling pathways both in vivo and in vitro. All the results suggested that peiminine exerted potent anti-inflammatory effects on LPS-induced mastitis in mice. Therefore, peiminine might be a potential therapeutic agent for mastitis.

Peiminine Protects Dopaminergic Neurons from Inflammation-Induced Cell Death by Inhibiting the ERK1/2 and NF-κB Signalling Pathways.

Neuroinflammation, characterized marked by microglial activation, plays a very important role in the pathogenesis of Parkinson's disease (PD). Upon activation, pro-inflammatory mediators are produced by microglia, triggering excessive inflammatory responses and ultimately damaging dopaminergic neurons. Therefore, the identification of agents that inhibit neuroinflammation may be an effective approach for developing novel treatments for PD. In this study, we sought to investigate whether peiminine protects dopaminergic neurons by inhibiting neuroinflammation. We evaluated the effects of peiminine on behavioural dysfunction, microglial activation and the loss of dopaminergic neurons in a rat model of lipopolysaccharide (LPS)-induced PD. BV-2 cells were pretreated with peiminine for 1 h and then stimulated with LPS for different times. Then, inflammatory responses and the related signalling pathways were analysed. Peiminine markedly attenuated behavioural dysfunction and inhibited the loss of dopaminergic neurons and microglial activation in the LPS-induced PD rat model. In BV-2 cells, peiminine significantly decreased LPS-induced expression of the pro-inflammatory mediators TNF-α, IL-6 and IL-1ß, COX-2 and iNOS by inhibiting the phosphorylation of ERK1/2, AKT and NF-κB p65. Based on these results demonstrated that peiminine has a role in protecting dopaminergic neurons in the LPS-induced PD rat model by inhibiting neuroinflammation.