[Effects of isopsoralen on tibial fracture and vascular healing in mice].

OBJECTIVE: To explore effects of isopsoralen (ISO) with different doses on fracture and vascular healing in mice. METHODS: Sixty 2-month-old male C57BL/6 mices with body mass of (20±2) g were selected and divided into 4 groups by random number table method:model group (model), low dose group (isopsoralen-low dose, ISO-L), medium dose group (isopsoralen-medium dose, ISO-M) and high dose group (isopsoralen-high dose, ISO-H), with 15 animals in each group. The right tibial fracture model was established. After operation, ISO-L group, ISO-M group and ISO-H group were given ISO concentration of 10 mg·kg-1, 20 mg·kg-1 and 40 mg·kg-1, respectively. Model group was given same volume of normal saline once a day for 28 days. Weighed once a week. X-ray was performed on 7, 14, 21 and 28 days, respectively, and modified I.R. Garrett scoring method was used to evaluate callus growth. After 28 days, the main organs were stripped and weighed, and organ coefficients were calculated. Hematoxylin eosin staining (HE staining) was performed on the organs to observe whether there were pathological structural changes. Micro-computed tomography (Micro-CT) was used to scan fracture area and conduct three-dimensional reconstruction to obtain the effect map, and quantify bone volume fraction (bone volume/total volume, BV/TV). After decalcification, the tibia was embedded in paraffin wax and sectioned. The healing and shape of fracture end were observed by HE staining and ferruxin solid green staining. The right tibia was removed and decalcified after intravascular infusion of Microfil contrast agent. Micro-CT was used to scan the callus microvessels in the fracture area, and the vascular volume fraction and vessel diameter were quantified. RESULTS: After 28 days of administration, there was no significant difference in body mass and organ coefficient among all groups (P>0.05), and no significant pathological changes were found in HE staining of organs. The results of X-ray and improved I.R. Garrett score showed that ISO-M group was higher than that of Model group at 28 days (P<0.05). Scores of ISO-H group at 14, 21 and 28 days were higher than those of the other 3 groups (P<0.05). Micro-CT results showed intracavitary callus in ISO-M group was significantly reduced, which was lower than that in Model group (P<0.05), most of the callus in ISO-H group were subsided, and BV/TV in ISO-H group was lower than that in the other 3 groups (P<0.05). The results of HE staining and ferrubens solid green staining showed fracture area of ISO-H group was closed, continuous laminar bone had appeared, and the fracture healing process was higher than that of other groups. Angiographic results showed vascular volume fraction in ISO-H and ISO-M groups was higher than that in Model and ISO-L groups (P<0.05), and the vascular diameter in ISO-H and ISO-M groups was higher than that in Model and ISO-L groups (P<0.05). CONCLUSION: In the concentration range of 10-40 mg·kg-1, ISO has no obvious toxic and side effects, and could improve bone microstructure, promote formation of callus microvessels, and accelerate healing of fracture ends in a concentration-dependent manner.

Mechanistic study of cytochrome P450 enzyme-mediated cytotoxicity of psoralen and isopsoralen.

Psoralen and isopsoralen are the major components responsible for Psoraleae Fructus-induced hepatotoxicity. This study explored the role of metabolic activation by cytochrome P450 (CYP) enzymes in psoralen- and isopsoralen-induced cytotoxicity and its potential mechanisms. Inhibitors of CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A4 were used to screen specific CYP enzymes responsible for the metabolic activation of psoralen and isopsoralen in mouse primary hepatocytes, which was verified using the corresponding transfected cell lines. Network toxicology and transcriptome analyses were performed to explore the mechanisms underlying toxicity. Psoralen and isopsoralen decreased the viability of mouse primary hepatocytes, whereas the inhibition of CYP2C9, 2C19, 2D6, and 2E1 significantly increased their viability. Psoralen-induced cytotoxicity was significantly enhanced by the overexpression of CYP2C19 in Chinese hamster ovary cells, whereas the overexpression of the above CYP enzymes did not affect the cytotoxicity of isopsoralen. Psoralen- and isopsoralen-induced cytotoxic effects were associated with putative core targets (i.e., Fn1, Thbs1, and Tlr2) and multiple signaling pathways (e.g., PI3K-Akt, MAPK, and TNF pathways). Our results demonstrate that the metabolic activation of psoralen and isopsoralen is mediated by CYP enzymes, thereby regulating multiple core targets and signaling pathways and resulting in cytotoxicity.

Isopsoralen promotes osteogenic differentiation of human jawbone marrow mesenchymal cells through Notch signaling pathway.

BACKGROUND: The aim of this study was to investigate the effect of isopsoralen on osteogenic differentiation of human jawbone marrow mesenchymal cells and its possible mechanism. METHOD: The cytotoxicity and proliferation of cells were measured by a cell counting kit 8. Alkaline phosphatase activity analysis was then used to determine the optimal concentration of isopsoralen to promote the differentiation. Western blot, qRT-PCR and Alizarin Red S staining were used to evaluate the role of Notch signaling pathway in isopsoralen-induced osteogenic differentiation. This study also investigated the anti-osteoporotic effects of ISO using in vivo osteoporosis models. RESULTS: Our results showed that 1 × 10-6 mol / L isopsoralen can effectively promote the proliferation and osteogenic differentiation of cells. Moreover, we found that activation of notch signaling pathway inhibited isopsoralen-induced osteogenesis and inhibition of Notch signal promoted the differentiation of osteoblasts induced by isopsoralen. In vivo experiments revealed that ISO significantly inhibited OVX-induced bone mineral density loss and restored the impaired bone structural properties in osteoporosis model mice. CONCLUSION: Our findings demonstrated that isopsoralen induced osteogenic differentiation by inhibiting Notch signaling and it might be a potential therapeutic agent for treating or preventing osteoporosis.

Unveiling hepatotoxicity distinction of coumarin-related compounds from glycosides to aglycones in Fructus Psoraleae by integrating UPLC-Q-TOF-MS and high content analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Fructus Psoraleae (FP), the dried and ripe fruit of Cullen corylifolium (L.) Medik., is widely used due to its various clinical pharmacological effects, but its hepatotoxicity restricts its clinical application. So far, its hepatotoxic components and their underlying mechanism have not been systematically elucidated. AIM OF THE STUDY: This study was undertaken to reveal the hepatotoxicity distinction of coumarin-related compounds from glycosides to aglycones in FP and elucidate their potential mechanism. METHODS: Rats were administrated with the aqueous extract of Fructus Psoraleae (AEFP), in which eight coumarin-related compounds were focused. Subsequently, compounds exposed in rats' livers were detected by UPLC-Q-TOF-MS, and the identified hepatotoxic compounds were evaluated to elaborate their possible mechanism by the aid of high content analysis (HCA). RESULTS: Eight coumarin-related compounds were identified, among which psoralenoside (PO), isopsoralenoside (IPO), psoralen (P), and isopsoralen (IP) were the principally exposed compounds in rats' livers. Furocoumarinic acid glucoside (FAG), (E)-3-(4-(((2S, 3R, 4S, 5S, 6R)-3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzofuran-5-yl) acrylic acid (isofurocoumarinic acid glucoside, IFAG), furocoumarinic acid (FA), and (E)-3-(4-hydroxybenzofuran-5-yl) acrylic acid (isofurocoumarinic acid, IFA) were also detected in low abundance. P, IP, FA, and IFA were identified as the hepatotoxic compounds, while their glycosides were almost non-hepatotoxic. The HCA's results showed that hepatotoxic compounds disrupted the balance in reactive oxygen species (ROS), nuclear area, and mitochondrial membrane potential of HepG2 cells, leading to the occurrence of hepatotoxicity. CONCLUSIONS: P, IP, FA, and IFA were identified as hepatotoxic compounds, from which P and IP were proposed as the important risk components for hepatotoxicity. The conversion from glycosides to aglycones played an essential role in FP-induced hepatotoxicity.

A study on the anti-osteoporosis mechanism of isopsoralen based on network pharmacology and molecular experiments.

OBJECTIVE: Osteoporosis (OP) is a disease caused by multiple factors. Studies have pointed out that isopsoralen (IPRN) is one of the most effective drugs for the treatment of OP. Based on network pharmacological and molecular experimental analysis, the molecular mechanism of IPRN in osteoporosis is clarified. METHODS: IPRN target genes and OP-related genes were predicted from the databases. Intersections were obtained and visualized. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on target genes, which was confirmed by experiments internal and external experiments. Molecular docking was used to verify the binding between IPRN and target proteins. Molecular dynamics (MD) simulates the binding affinity of protein targets and active compounds. RESULTS: 87 IPRN target genes and 242 disease-related targets were predicted. The protein-protein interaction (PPI) network identified 18 IPRN target proteins for the treatment of OP. GO analysis indicated that target genes were involved in biological processes. KEGG analysis showed that pathways such as PI3K/AKT/mTOR were associated with OP. Cell experiments (qPCR and WB) found that the expressions of PI3K, AKT, and mTOR in MC3T3-E1 cells at 10 µM, 20 µM, and 50 µM IPRN concentrations, especially at 20 µM IPRN treatment, were higher than those in the control group at 48 h. Animal experiments also showed that compared with the control group, 40 mg/kg/time IPRN could promote the expression of the PI3K gene in chondrocytes of SD rats. CONCLUSIONS: This study predicted the target genes of IPRN in the treatment of OP and preliminarily verified that IPRN plays an anti-OP role through the PI3K/AKT/mTOR pathway, which provides a new drug for the treatment of OP.

Isopsoralen suppresses receptor activator of nuclear factor kappa-ß ligand-induced osteoclastogenesis by inhibiting the NF-κB signaling.

Osteoporosis is a serious systemic metabolic bone system disease.This study aimed to identify the target genes of isopsoralen and the signaling pathways involved in the differential expression of the genes involved in osteoclast differentiation. We hypothesized that isopsoralen may inhibit osteoclast differentiation by blocking the nuclear factor kappa-B (NF-κB) signaling pathway and verified our hypothesis through basic experiments. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to detect the effect of isopsoralen on the proliferation and viability of primary mouse bone marrow monocytes (BMMCs). The effect of isopsoralen on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation was determined by using tartrate-resistant acid phosphatase (TRAP) staining. Quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the expression of the related genes and proteins. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of isopsoralen target genes were obtained through comprehensive analysis using the STITCH database, Cytoscape 3.8.2, and R-Studio software. Differentially expressed genes (DEGs) were found in osteoclasts induced by RANKL before and after 3 days using R-Studio, following which KEGG analysis was performed. Next, enrichment analysis was performed on the KEGG pathway shared by the target genes of isopsoralen and the differentially expressed genes during osteoclast differentiation to predict the signaling pathway underlying the inhibition of osteoclast differentiation by isopsoralen. Finally, Western blot was used to detect the effect of isopsoralen on the activation of signaling pathways to verify the results of our bioinformatics analysis. Based on the enrichment analysis of isopsoralen target genes and differentially expressed genes during osteoclastogenesis, we believe that isopsoralen can inhibit RANKL-induced osteoclastogenesis by inhibiting the NF-κB signaling pathway.

Transcriptomics and metabolomics reveal the role of CYP1A2 in psoralen/isopsoralen-induced metabolic activation and hepatotoxicity.

Psoralen and isopsoralen are the pharmacologically important but hepatotoxic components in Psoraleae Fructus. The purpose of this study was to reveal the underlying mechanism of psoralen/isopsoralen-induced hepatotoxicity. Initially, we applied integrated analyses of transcriptomic and metabolomic profiles in mice treated with psoralen and isopsoralen, highlighting the xenobiotic metabolism by cytochromes P450 as a potential pathway. Then, with verifications of expression levels by qRT-PCR and western blot, affinities by molecular docking, and metabolic contributions by recombinant human CYP450 and mouse liver microsomes, CYP1A2 was screened out as the key metabolic enzyme. Afterwards, CYP1A2 induction and inhibition models in HepG2 cells and mice were established to verify the role of CYP1A2, demonstrating that induction of CYP1A2 aggravated the hepatotoxicity, and conversely inhibition alleviated the hepatotoxic effects. Additionally, we detected glutathione adducts with reactive intermediates of psoralen and isopsoralen generated by CYP1A2 metabolism in biosystems of recombinant human CYP1A2 and mouse liver microsomes, CYP1A2-overexpressed HepG2 cells, mice livers and the chemical reaction system using UPLC-Q-TOF-MS/MS. Ultimately, the high-content screening presented the cellular oxidative stress and relevant hepatotoxicity due to glutathione depletion by reactive intermediates. In brief, our findings illustrated that CYP1A2-mediated metabolic activation is responsible for the psoralen/isopsoralen-induced hepatotoxicity.

Anti-Postmenopausal osteoporosis effects of Isopsoralen: A bioinformatics-integrated experimental study.

Isopsoralen (IPRN), which comes from the fruit of Psoralea corylifolia, has been identified as a kind of phytoestrogen and has been proven to be effective for the treatment of osteoporosis (OP). However, the mechanisms underlying IPRN's anti-OP effects, especially the anti-postmenopausal osteoporosis (PMOP) effects, remain indistinct. Thus, this study aimed to investigate the effects and mechanisms of IPRN's anti-PMOP activity. In this study, the bioinformatics results predicted that IPRN could resist PMOP by targeting EGFR, AKT1, SRC, CCND1, ESR1 (ER-α), AR, PGR, BRCA1, PTGS2, and IGF1R. An ovariectomized (OVX) mice model and a H2 O2 -induced bone marrow mesenchyml stem cells (BMSCs) model confirmed that IPRN could inhibit the bone loss induced by OVX in mice and promote the osteogenic differentiation in H2 O2 -induced BMSCs by inhibiting oxidative stress and apoptosis. Moreover, IPRN could significantly produce the above effects by upregulating ESR1. IPRN might be a therapeutic agent for PMOP by acting as an estrogen replacement agent and a natural antioxidant.

Effects of isopsoralen on tibial fracture and vascular healing in mice / 中国骨伤

OBJECTIVE@#To explore effects of isopsoralen (ISO) with different doses on fracture and vascular healing in mice.@*METHODS@#Sixty 2-month-old male C57BL/6 mices with body mass of (20±2) g were selected and divided into 4 groups by random number table method:model group (model), low dose group (isopsoralen-low dose, ISO-L), medium dose group (isopsoralen-medium dose, ISO-M) and high dose group (isopsoralen-high dose, ISO-H), with 15 animals in each group. The right tibial fracture model was established. After operation, ISO-L group, ISO-M group and ISO-H group were given ISO concentration of 10 mg·kg-1, 20 mg·kg-1 and 40 mg·kg-1, respectively. Model group was given same volume of normal saline once a day for 28 days. Weighed once a week. X-ray was performed on 7, 14, 21 and 28 days, respectively, and modified I.R. Garrett scoring method was used to evaluate callus growth. After 28 days, the main organs were stripped and weighed, and organ coefficients were calculated. Hematoxylin eosin staining (HE staining) was performed on the organs to observe whether there were pathological structural changes. Micro-computed tomography (Micro-CT) was used to scan fracture area and conduct three-dimensional reconstruction to obtain the effect map, and quantify bone volume fraction (bone volume/total volume, BV/TV). After decalcification, the tibia was embedded in paraffin wax and sectioned. The healing and shape of fracture end were observed by HE staining and ferruxin solid green staining. The right tibia was removed and decalcified after intravascular infusion of Microfil contrast agent. Micro-CT was used to scan the callus microvessels in the fracture area, and the vascular volume fraction and vessel diameter were quantified.@*RESULTS@#After 28 days of administration, there was no significant difference in body mass and organ coefficient among all groups (P>0.05), and no significant pathological changes were found in HE staining of organs. The results of X-ray and improved I.R. Garrett score showed that ISO-M group was higher than that of Model group at 28 days (P<0.05). Scores of ISO-H group at 14, 21 and 28 days were higher than those of the other 3 groups (P<0.05). Micro-CT results showed intracavitary callus in ISO-M group was significantly reduced, which was lower than that in Model group (P<0.05), most of the callus in ISO-H group were subsided, and BV/TV in ISO-H group was lower than that in the other 3 groups (P<0.05). The results of HE staining and ferrubens solid green staining showed fracture area of ISO-H group was closed, continuous laminar bone had appeared, and the fracture healing process was higher than that of other groups. Angiographic results showed vascular volume fraction in ISO-H and ISO-M groups was higher than that in Model and ISO-L groups (P<0.05), and the vascular diameter in ISO-H and ISO-M groups was higher than that in Model and ISO-L groups (P<0.05).@*CONCLUSION@#In the concentration range of 10-40 mg·kg-1, ISO has no obvious toxic and side effects, and could improve bone microstructure, promote formation of callus microvessels, and accelerate healing of fracture ends in a concentration-dependent manner.

Psoralen and isopsoralen from Psoraleae Fructus aroused hepatotoxicity via induction of aryl hydrocarbon receptor-mediated CYP1A2 expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Psoraleae Fructus (PF), a traditional Chinese medicine, has long been used to treat diseases such as cancer, osteoporosis and leukoderma. Psoralen and isopsoralen are main bioactive ingredients of PF with anti-tumor, anti-inflammatory, estrogen-like neuroprotection, etc., meanwhile they are also representative hepatotoxic components of PF. Hepatic CYP1A2 has been reported to be the important metabolic enzymes involved in psoralen and isopsoralen-induced hepatotoxicity. However, the relationship between the hepatotoxicity and CYP1A2 expression, and the underlying mechanism of regulating CYP1A2 expression remain unclear. AIM OF STUDY: The aim of this study was to explore the associated mechanism between psoralen or isopsoralen induced hepatotoxicity and activated aryl hydrocarbon receptor (AhR)-mediated transcriptional induction of CYP1A2 in vitro and in vivo. MATERIALS AND METHODS: Psoralen and isopsoralen at different doses were treated on HepG2 cells (10, 25, 50, 100, 200 µM for 2, 12, 24, 36, 48 h) and mice (20, 80, 160 mg/kg for 3, 7, 14 days) for different time, to assess the correlation of induced hepatotoxicity and CYP1A2 mRNA and protein expression in vivo and in vitro, as well as the effect on CYP1A2 enzyme activity evaluated by phenacetin metabolism. In addition, the potential mechanism of the regulation of CYP1A2 expression mediated by AhR was explored through nucleocytoplasmic shuttling, immunofluorescence, cellular thermal shift assay and molecular docking, etc. RESULTS: Psoralen and isopsoralen induced cytotoxicity in HepG2 cells, and hepatomegaly, biochemicals disorder and tissue pathological impairment in mice, respectively in dose- and time-dependent manners. Simultaneously accompanied with elevated levels of CYP1A2 mRNA and protein in the same trend, and the CYP1A2 activity was remarkably inhibited in vitro but significantly elevated overall in vivo. Besides, psoralen and isopsoralen bound to AhR and activated translocation of AhR from the cytoplasm to the nucleus, leading to the transcriptional induction of target gene CYP1A2. CONCLUSIONS: Hepatotoxicities in HepG2 cells and mice aroused by psoralen and isopsoralen were related to the induction of CYP1A2 expression and activity, whose underlying mechanism might be psoralen or isopsoralen activated AhR translocation and induced increase of CYP1A2 transcriptional expression. Hopefully, these finding are conductive to propose an alert about the combined usage of psoralen or isopsoralen and AhR ligands or CYP1A2 substrates in clinical practice.

[Biotransformation of psoralenoside and isopsoralenoside in Psoraleae Fructus incubated with human intestinal bacteria flora in vitro].

Absorption is crucial to the resultant efficacy of oral drugs where the intestinal bacteria flora functions as one of the first-pass effects.The present study investigated the biotransformation of psoralenoside and isopsoralenoside in Chinese medicine Psoraleae Fructus(the dried fruit of Psoralea corylifolia) with the internationally recognized human intestinal bacteria flora model in vitro.Pso-ralenoside and isopsoralenoside were anaerobically incubated with human intestinal bacteria flora at 37 ℃, respectively, and biotransformation products were analyzed and identified using high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS) and comparison with reference standards.The main biotransformation products of psoralenoside were psoralen and a small amount of 6,7-furano-hydrocoumaric acid, and the main biotransformation products of isopsoralenoside were isopsoralen and a small amount of 5,6-furano-hydrocoumaric acid.

Protective effect of isopsoralen on UVB-induced injury in HaCaT cells via the ER and p38MAPK signaling pathways.

This study investigated the protective effect of isopsoralen on UVB-induced damage in HaCaT cells and its molecular mechanism. The cytotoxicity of isopsoralen and its effects on the viability of HaCaT cells were examined using the MTT assay. The effects of UVB irradiation and isopsoralen on the intracellular glutathione (GSH-PX), superoxide dismutase (SOD), malondialdehyde (MDA), and reactive oxygen species (ROS) content were examined using commercially available assay kits. Further, the effects of UVB irradiation and isopsoralen on the levels of the inflammatory cytokines TNF-α, IL-6, and IL-1α were examined using enzyme-linked immunosorbent assay. Finally, we examined the effect of adding the estrogen receptor (ER) antagonist ICI182780,780 and the p38MAPK antagonist SB203580 on the changes in inflammatory cytokines induced by isopsoralen treatment and UVB irradiation. Isopsoralen pretreatment markedly inhibited UVB-induced reduction in the viability and proliferation of HaCaT cells. Isopsoralen also reduced UVB-induced increase in the expression of the inflammatory cytokines and the level of free radicals (ROS and MDA), and reversed the UVB-induced suppression of antioxidant activity. Additionally, inhibition of ER and p38MAPK via the addition of their respective antagonists reversed the observed anti-inflammatory effects of Isopsoralen. Isopsoralen can efficiently provide protection against UVB-induced damage in HaCaT cells brought about via oxidation and inflammatory reactions, and the underlying mechanisms involve the ER and p38MAPK pathways. Therefore, Isopsoralen could be used in therapeutic solutions for UVB-induced skin conditions. PRACTICAL APPLICATIONS: Isopsoralen shows antioxidant and anti-inflammatory effects. As natural, healthy, and effective additives, isopsoralen has been widely used in cosmetics and botanical medicine products. The results of this study reveal the molecular mechanisms underlying isopsoralen effects, showing that isopsoralen reverses the effects of UVB irradiation regulating ER and p38MAPK signaling pathways. Consequently, isopsoralen regulates the expression of ER and p38MAPK signaling pathways, thereby reducing the activation of antioxidant and anti-inflammatory activity. These findings suggest that isopsoralen can be used as the base ingredient for antiphotoaging cosmetics and botanical medicine products. This study provides both theoretical and experimental background for isopsoralen deep processing and utilization.

Isopsoralen ameliorates rheumatoid arthritis by targeting MIF.

BACKGROUND: Isopsoralen (IPRN), one of the active ingredients of Psoralea corylifolia Linn, has anti-inflammatory properties. We attempted to investigate the inhibitory effects of IPRN on rheumatoid arthritis (RA) and characterize its potential mechanism. METHODS: RA fibroblast-like synoviocytes (FLSs) and mice with collagen-induced arthritis (CIA) were used as in vitro and in vivo models to analyze the antiarthritic effect of IPRN. Histological analysis of the inflamed joints from mice with CIA was performed using microcomputed tomography (micro-CT) and hematoxylin-eosin (HE) staining. RNA sequencing (RNA-Seq), network pharmacology analysis, molecular docking, drug affinity responsive target stability (DARTS) assay, and cellular thermal shift assay (CETSA) were performed to evaluate the targets of IPRN. RESULTS: IPRN ameliorated the inflammatory phenotype of RA FLSs by inhibiting their cytokine production, migration, invasion, and proangiogenic ability. IPRN also significantly reduced the severity of CIA in mice by decreasing paw thickness, arthritis score, bone damage, and serum inflammatory cytokine levels. A mechanistic study demonstrated that macrophage migration inhibitory factor (MIF), a key protein in the inflammatory process, was the specific target by which IPRN exerted its anti-inflammatory effects in RA FLSs. CONCLUSION: Our study demonstrates the antiarthritic effect of IPRN, which suggests the therapeutic potential of IPRN in RA.

Enhancement of autophagy flux by isopsoralen ameliorates interleukin-1ß-stimulated apoptosis in rat chondrocytes.

Chondrocyte apoptosis contributes to the pathogenesis of cartilage degeneration in osteoarthritis (OA). We found that isopsoralen pretreatment significantly reversed the increase in DNA fragmentation and apoptosis rate, and significantly decreased the caspase-3 activity and PARP cleavage in IL-1ß-treated chondrocytes. Isopsoralen pretreatment markedly inhibited disruption of matrix proteins. Moreover, the expressions of LC3-II and LAMP-1 were markedly increased but the expression of p62/SQSTM1 was remarkably decreased by isopsoralen pretreatment. Importantly, the protective effects of isopsoralen against IL-1ß were blocked by pretreatment with autophagy inhibitor 3-MA and bafilomycin A1. These results suggest that isopsoralen ameliorates chondrocyte apoptosis by promoting autophagy flux.[Formula: see text].

Transformation of Psoralen and Isopsoralen by Human Intestinal Microbial In Vitro, and the Biological Activities of Its Metabolites.

Psoralen (P) and isopsoralen (IP) are the main active ingredients in the dried fruit of Psoralen corylifolia L. (PC), with a wide range of pharmacology activities. The intestinal bacteria biotransformation plays a central role in the metabolism of the complex ingredients in traditional Chinese medicine (TCM). Our study aimed to investigated the metabolic profile of P and IP in the intestinal condition, co-cultured with human fecal bacteria anaerobically. Four bio-transforming products were obtained, including 6,7-furano-hydrocoumaric acid (P-1) and 6,7-furano-hydro- coumaric acid methyl ester (P-2), which transformed from P, and 5,6-furano-hydrocoumaric acid (IP-1) and 5,6-furano-hydrocoumaric acid methyl ester (IP-2), which were transformed from IP. It is worth mentioning that IP-2 is a new compound that has not been published. Their structures were analyzed based on their spectroscopic data. Moreover, a highly sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was used to characterize the metabolic pathways of P, IP, and their bio-transforming products in the reaction samples. In addition, the dampening effects against the oxidative stress of P, IP, and their bio-transforming products by human intestinal flora were estimated in vitro via the human colorectal cells (HCT116) and heterogeneous human epithelial colorectal adenocarcinoma cells (Caco-2) cell lines. The results showed that the metabolites have stronger activity than P and IP, which possibly provides a basis for elucidating the treating mechanisms of PC extract against inflammatory bowel disease.

Long-Term Exposure of Psoralen and Isopsoralen Induced Hepatotoxicity and Serum Metabolites Profiles Changes in Female Rats.

:Pre-clinical safety evaluation of traditional medicines is imperative because of the universality of drug-induced adverse reactions. Psoralen and isopsoralen are the major active molecules and quality-control components of a traditional herbal medicine which is popularly used in Asia, Fructus Psoraleae. The purpose of this study is to assess the long-term effects of psoralen and isopsoralen with low levels on the biochemical parameters and metabolic profiles of rats. Three doses (14, 28, and 56 mg/kg) of psoralen and one dose (28 mg/kg) of isopsoralen were administered to rats over 12 weeks. Blood and selected tissue samples were collected and analyzed for hematology, serum biochemistry, and histopathology. Metabolic changes in serum samples were detected via proton nuclear magnetic resonance (1H-NMR) spectroscopy. We found that psoralen significantly changed the visceral coefficients, blood biochemical parameters, and histopathology, and isopsoralen extra influenced the hematological index. Moreover, psoralen induced remarkable elevations of forvaline, isoleucine, isobutyrate, alanine, acetone, pyruvate, glutamine, citrate, unsaturated lipids, choline, creatine, phenylalanine, and 4-hydroxybenzoate, and significant reductions of ethanol and dimethyl sulfone. Isopsoralen only induced a few remarkable changes of metabolites. These results suggest that chronic exposure to low-level of psoralen causes a disturbance in alanine metabolism, glutamate metabolism, urea cycle, glucose-alanine cycle, ammonia recycling, glycine, and serine metabolism pathways. Psoralen and isopsoralen showed different toxicity characteristics to the rats.

Isopsoralen ameliorates H2O2-induced damage in osteoblasts via activating the Wnt/ß-catenin pathway.

Osteoporosis is a disease with a worldwide prevalence that involves a severe loss of bone mineral density and decreased microarchitecture, which increases the risk of bone fracture. The present study evaluated the effects of isopsoralen on osteoblastic OB-6 cells following hydrogen peroxide (H2O2)-induced damage and investigated the molecular mechanisms involved in this process. For in vitro experiments, OB-6 osteoblasts were treated with H2O2 or H2O2 + isopsoralen then the cell viability, apoptosis, reactive oxygen species (ROS) production and calcium accumulation were determined. Results demonstrated that treatment with H2O2 reduced cell viability, runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) expression levels, and calcium deposition, whilst markedly increasing cell apoptosis and ROS production. However, isopsoralen (1 µM) provided significant protection against H2O2-induced alterations in osteoblasts. In addition, isopsoralen effectively upregulated protein expression of tankyrase and ß-catenin which are the main transductors of the Wnt/ß-catenin pathway. Of note, the protective effects of isopsoralen against H2O2-induced damage were attenuated in OB-6 cells treated with tankyrase inhibitor XAV-939. In conclusion, the present findings provided evidence that isopsoralen attenuated oxidative stress-induced injury in osteoblasts via the Wnt/ß-catenin signaling pathway.

[Analysis of standard decoction of salt-processed Psoraleae Fructus].

The traditional Chinese medicine standard decoction is prepared on the basis of the theory of traditional Chinese medicine and clinical application. With reference to the modern extraction method,the single decoction of traditional Chinese medicine is prepared by the standardized process,and the establishment of its quality standards is conducive to standardizing clinical medication. This research is to set an evaluation standard for the quality of salt-processed Psoraleae Fructus standard decoction. Twelve batches of salt-processed Psoraleae Fructus standard decoctions were prepared. The contents of psoralen and isopsoralen were determined,the transfer and extract rates were calculated,and the pH value was measured; HPLC fingerprint method was established for analysis. The results of the 12 batches of samples revealed that the transfer rates of psoralen and isopsoralen were 17. 10%-26. 40%,14. 70%-22. 70%,respectively; the extract rate was between 14. 7%-27. 0%,and the pH value was between 5. 4-6. 9. Moreover,7 common chromatographic peaks were determined based on fingerprint by using similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine( 2012 A).The similarities of the 12 batches of samples were analyzed and compared,and the results showed that the similarities were all higher than0. 9. In this study,the preparation method for salt-processed Psoraleae Fructus decoction was standard,with high similarities in fingerprint. This study build a convenient and reliable method of comprehensive quality evaluation,with a high precision,stability and repeatability,which can provide a reference for the quality control of salt-processed Psoraleae Fructus dispensing granules.

[Psoralen and isopsoralen improve lipid metabolism disorder via inhibition of NF-κB activation in LO2 cells].

The aim of this paper was to investigate the mechanism and effect of psoralen and isopsoralen in the treatment of lipid accumulation in LO2 cells. Human LO2 cells nonalcoholic fatty liver models were established by using palmitic acid( PA). Then psoralen and isopsoralen were administered for intervention. Intracellular triglyceride( TG) and total cholesterol( TC) content,the cell supernatant alanine aminotransferase( ALT) and aspartate aminotransferase( AST) levels were determined by enzyme method. Cell supernatant proinflammatory cytokines( IL-6,TNF-α) and chemokines( IL-8,MCP-1) were determined by ELISA method. Western blot method was conducted to detect the protein expression of intracellular nuclear factor( NF-κB) p65 phosphorylation( p-p65),nonphosphorylated protein( p65),and transforming factor TGF-ß1. Result showed that as compared with the model group,intracellular TG and TC levels,the cell supernatant ALT and AST levels,proinflammatory cytokines and chemokines were decreased( P < 0. 01,P <0. 05); the p-p65/p65 ratio and TGF-ß1 protein expression were also significantly decreased( P< 0. 01,P< 0. 05) in psoralen intervention group. As compared with the model cells,intracellular TG content had no significant changes,but all the other indexes were reduced( P<0. 01,P<0. 05) in the cells of isopsoralen intervention group. Psoralen exhibited better effect than isopsoralen( P< 0. 01,P<0. 05). It is concluded that psoralen could improve the adipogenesis of LO2 cells induced by PA; both psoralen and isopsoralen are effective in ameliorating LO2 cells injury induced by PA,reducing inflammation via inhibiting the activation of NF-κB and down-regulating the expression of TGF-ß1.

The mechanism of Psoralen and Isopsoralen hepatotoxicity as revealed by hepatic gene expression profiling in SD rats.

BACKGROUND: The main bioactive components of Fructus psoraleae, such as psoralen and isopsoralen, are known to be hepatotoxic. However, its underlying mechanism is to be elucidated. METHODS: To address this, SD rats were randomly divided into control group, 60 mg/kg psoralen group and 60 mg/kg isopsoralen group. Blood was collected to detect serum biochemical indices. RNA was extracted from liver samples, and then, cDNA gene expression profiles were analysed. RESULTS: Psoralen administration significantly up-regulated serum AST (aspartate aminotransferase) while addition of isopsoralen increased serum ALT (alanine aminotransferase), AST, TBA (total bile acid) and TG (total triglyceride) levels. A total of 172 differentially expressed genes (DEGs) were acquired between psoralen group and control group while 884 DEGs were screened between isopsoralen group and control group. Chemical Carcinogenesis and Metabolism of Xenobiotics by Cytochrome P450 were the two most significantly enriched pathways as revealed by DEGs. Liver was the most impacted organ, and endoplasmic reticulum was the most impacted organelle in subcellular level. Finally, some kinds of cancers and cytochrome p450 oxidoreductase deficiency were predicted. Taken together, psoralen and isopsoralen might cause hepatotoxicity mainly through cytochrome P450 metabolism of xenobiotics. Furthermore, Cyp1a1, Cyp1a2, Gstm1 and Akr7a3 worked as key genes in hepatotoxicity. Moreover, endoplasmic reticulum was the main target subcellular structure in hepatotoxicity induced by psoralen and isopsoralen.