Sophocarpine Research Articles

Sophocarpine attenuates doxorubicin-induced heart injury through inhibition of fibrosis.

Doxorubicin (DOX) is a potent anti-cancer medication that is associated with numerous adverse effects, particularly concerning damage to the heart. This study aimed to investigate the impact of sophocarpine (SOP) on DOX-induced heart injury through both in vivo and in vitro experiments. The experimental techniques employed encompassed echocardiography, hematoxylin/eosin (H&E) staining, Masson staining, immunohistochemical staining, western blotting, and so on. Echocardiography showed that SOP alleviated DOX-induced cardiac dysfunction, as evidenced by the improvements in both left ventricle ejection fraction and left ventricle fractional shortening. DOX caused upregulations of creatine kinase-MB and lactate dehydrogenase, while SOP decreased these indices. Staining methods such as H&E and Masson showed that SOP reversed the pathological changes induced by DOX. DOX elevated the expression levels of fibrosis-associated proteins such as Collagen I, Collagen III, α-SMA, Fibronectin, MMP-2, and MMP-9. However, SOP reversed these changes. Moreover, the study further revealed that SOP inhibited the TGF-β1/Smad3 signaling pathway. These findings imply that SOP has the potential to mitigate DOX-induced heart injury by suppressing fibrosis. The underlying molecular mechanism may involve the inhibition of the TGF-β1/Smad3 signaling pathway.

Sophocarpine alleviates doxorubicin-induced heart injury by suppressing oxidative stress and apoptosis

Doxorubicin (DOX) is an effective anti-tumor drug accompanied with many side effects, especially heart injury. To explore what effects of sophocarpine (SOP) on DOX-induced heart injury, this study conducted in vivo experiment and in vitro experiment, and the C57BL/6J mice and the H9C2 cells were used. The experimental methods used included echocardiography, enzyme-linked immunosorbent assay (ELISA), dihydroethidium (DHE) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, western blotting and so on. Echocardiography showed that SOP alleviated DOX-induced cardiac dysfunction, as evidenced by the improvements of left ventricle ejection fraction and left ventricle fractional shortening. DOX caused upregulations of creatine kinase (CK), creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH), while SOP reduced these indices. The relevant stainings showed that SOP reversed the increases of total superoxide level induced by DOX. DOX also contribute to a higher level of MDA and lower levels of SOD and GSH, but these changes were suppressed by SOP. DOX increased the pro-oxidative protein level of NOX-4 while decreased the anti-oxidative protein level of SOD-2, but SOP reversed these effects. In addition, this study further discovered that SOP inhibited the decreases of Nrf2 and HO-1 levels induced by DOX. The TUNEL staining revealed that SOP reduced the high degree of apoptosis induced by DOX. Besides, pro-apoptosis proteins like Bax, cleaved-caspase-3 and cytochrome-c upregulated while anti-apoptosis protein like Bcl-2 downregulated when challenged by DOX, but them were suppressed by SOP. These findings suggested that SOP could alleviate DOX-induced heart injury by suppressing oxidative stress and apoptosis, with molecular mechanism activating of the Nrf2/HO-1 signaling pathway.

Sophocarpine Alleviates Isoproterenol-Induced Kidney Injury by Suppressing Inflammation, Apoptosis, Oxidative Stress and Fibrosis.

One of the most common diseases affecting people and leading to high morbidity is kidney injury. The alleviation of inflammation and apoptosis is considered a potential therapeutic approach for kidney injury. Sophocarpine (SOP), a tetracyclic quinolizidine alkaloid, exhibits various beneficial biological properties. To investigate the effects of SOP on isoproterenol (ISO)-induced kidney injury, we randomly divided mice into four groups: Control, ISO, ISO+SOP (20 mg/kg) and ISO+SOP (40 mg/kg). SOP was administered intraperitoneally to the mice over two weeks, accompanied by intraperitoneal stimulation of ISO (10 mg/kg) for another four weeks. After the mice were sacrificed, several methods such as ELISA, staining (H&E, TUNEL, DHE and Masson) and Western blotting were applied to detect the corresponding indicators. The kidney injury serum biomarkers SCr and BUN increased after the ISO challenge, while this effect was reversed by treatment with SOP. Pathological changes induced by ISO were also reversed by treatment with SOP in the staining. The inflammatory cytokines IL-β, IL-6, TNF-α, MCP-1 and NLRP3 increased after the challenge with ISO, while they were decreased by treatment with SOP. The apoptotic proteins cleaved-caspase-3 and Bax increased, while Bcl-2 decreased, after the challenge with ISO, and these effects were reversed by treatment with SOP. The antioxidant proteins SOD-1 and SOD-2 decreased after being stimulated by ISO, while they increased after the treatment with SOP. The fibrotic proteins collagen I, collagen III, α-SMA, fibronectin, MMP-2 and MMP-9 increased after the challenge with ISO, while they decreased after the treatment with SOP. We further discovered that the TLR-4/NF-κB and TGF-β1/Smad3 signaling pathways were suppressed, while the Nrf2/HO-1 signaling pathway was activated. In summary, SOP could alleviate ISO-induced kidney injury by inhibiting inflammation, apoptosis, oxidative stress and fibrosis. The molecular mechanisms were suppression of the TLR-4/NF-κB and TGF-β1/Smad3 signaling pathways and activation of the Nrf2/HO-1 signaling pathway, indicating that SOP might serve as a novel therapeutic strategy for kidney injury.

Network pharmacology and experimental validation identify the potential mechanism of sophocarpine for COVID-19.

Introduction. Coronavirus disease 2019 (COVID-19) has caused a serious threat to public health worldwide, and there is currently no effective therapeutic strategy for treating COVID-19.Hypothesis/Gap Statement. We propose that sophocarpine (SOP) might have potential therapeutic effects on COVID-19 through inhibiting the cytokine storm and the nuclear factor NF-κB signalling pathway.Aim. The objective was to elucidate the potential mechanism of SOP against COVID-19 through a network pharmacology analysis and its experimental validation.Methodology. The BATMAN-TCM database was used to identify the therapeutic targets of SOP, while the GeneCards and DisGeNET databases were used to identify the targets related to COVID-19. A protein-protein interaction (PPI) network was constructed from the STRING and analysed using Cytoscape software. Gene ontology (GO), Kyoto Encyclopaedia of Genes and Genomes (KEGG) and disease ontology (DO) enrichment analyses of the co-targets were performed using Metascape. Autodock 4.2.6 and Pymol software were applied for molecular docking. Levels of the proinflammatory cytokines IL-6, TNFα and IL-1β were measured by ELISA, while mRNA expression levels of intercellular adhesion molecule 1 (ICAM-1), vascular endothelial growth factor A (VEGFA) and IFN gamma (IFNG) were detected by real-time quantitative reverse transcription PCR. The protein levels of the molecules involved in the NF-κB signalling pathway were validated by western blot analysis.Results. A total of 65 co-targets of SOP and COVID-19 were determined. GO and KEGG enrichment analyses suggested that SOP affected COVID-19 by regulating the IL-17 signalling pathway, TNF signalling pathway and other signalling pathways. The PPI network and molecular docking showed that p65, ICAM-1 and VEGFA were key targets of SOP against COVID-19 and the underlying mechanism was validated in A549 cells in vitro. SOP attenuated the LPS-induced production of TNF-α and IL-6 and downregulated the LPS-induced mRNA expression of ICAM-1, VEGFA and IFNG. Mechanistically, SOP pretreatment inhibited the phosphorylation of p65 and facilitated the activation of Nrf2.Conclusions. SOP has a potential therapeutic effect on COVID-19 through multiple pathways and targets, and inhibits the production of pro-inflammatory cytokines and molecules involved in the NF-κB signalling pathway.

Sophocarpine Alleviates Injury-Induced Intima Hyperplasia of Carotid Arteries by Suppressing Inflammation in a Rat Model.

Introduction: Balloon angioplasty is a commonly applied procedure for treating atherosclerotic vascular diseases. However, the maintenance of long-term lumen patency is relatively difficult due to the occurrence of restenosis. Previous research has shown that the occurrence of vascular wall inflammation is associated with higher rates of restenosis. Sophocarpine (SPC) can exert various therapeutic effects such as anti-oxidation, anti-inflammation, anti-tumor, antivirus and immune regulation. This study aimed to investigate whether SPC can alleviate intimal hyperplasia following balloon injury in a rat carotid artery model. Methods: Twenty Sprague–Dawley rats were randomly assigned to four groups: (i) control, (ii) balloon injury, (iii) balloon injury followed by saline injection, and (iv) balloon injury followed by SPC administration. Each group contained five rats. A high-pressure balloon of 3 mm × 20 mm was placed in the carotid artery. The balloon was inflated to a pressure of 8 atmospheres to carry out rat carotid artery balloon injury model. The areas of neointimal and media were determined by Verhoeff_Van Gieson staining, and the intima-to-media (I:M) ratios were subsequently evaluated. After that, the protein levels of IL-6, IL-1β, MCP-1, NF-κB, TNF-α, VCAM-1, ICAM-1 and eNOS were measured. Results: The ratio of I:M was remarkably higher in the balloon injury group than in the control group (p < 0.01). SPC could significantly decrease the ratio of I:M compared with the balloon injury group (p < 0.01). Besides, the protein levels of IL-6, IL-1β, MCP-1, NF-κB, TNF-α, ICAM-1 and VCAM-1 were increased in rat carotid arteries exposed to balloon injury (p < 0.01), and treatment with SPC could attenuate these effects (p < 0.05). Furthermore, balloon injury inhibited the protein expression of eNOS (p < 0.01), and SPC could elevate its level (p < 0.05). Conclusions: SPC could alleviate an intimal hyperplasia in balloon-injured carotid artery, and the mechanisms underlying this protective effect might be due to its inhibitory potency against inflammation signals. Our study also implies the potential applicability of SPC in treating restenosis after balloon angioplasty.

Proteome profiling reveals the efficacy and targets of sophocarpine against asthma

Sophocarpine (SPC) as a quinolizidine alkaloid displays powerful effects on inflammatory diseases through regulating multiple targets. Asthma is a complex heterogeneous and inflammatory disease with an increasing incidence worldwide. Here we established a mice asthma model and investigated the effect of SPC. Mice induced by ovalbumin (OVA) exhibits exacerbated Th1/Th2 immune imbalance and allergic lung inflammation. SPC treatment regulated Th1/Th2 cytokines production (IL-4, IL-5 and INF-γ) in BALF, reduced IgE level in serum, inhibited inflammatory cell infiltration, and improved the lung tissue pathology. Proteomic results showed that 5064 proteins in lung tissue were detected and among them 223 preliminary therapeutic targets of SPC were selected. Subsequently, excluding non-human genes, 109 targets with established crystal structures were harvested. Meanwhile, the molecular docking results showed that the binding energy of 87 targets with SPC was varied from −9.72 kcal/mol to 227.16 kcal/mol. Further, SPC suppressed arrb2, anxa1, myd88 and sphk1 expression and activated p-stat1. All of the five targets based on the screened results of proteomics and molecular docking are critical in allergic asthma. Thus, our data revealed that SPC alleviated bronchial asthma via targeting multi-targets.

Sophocarpine can enhance the inhibiting effect of oxaliplatin on colon cancer liver metastasis-in vitro and in vivo.

To determine whether sophocarpine (SC) can enhance the inhibiting effect of oxaliplatin on colorectal cancer (CRC) liver metastasis (CRCLM)-in vitro and in vivo. The inhibitory effect of SC and/or oxaliplatin on the proliferation, invasion, and migration of CRC cells was determined by CCK-8, MTT, wound healing migration, and transwell assay. Western blot and immunohistochemistry were used to evaluate the inhibitory effect of SC and/or oxaliplatin on the epithelial-mesenchymal transition (EMT) process. Nude mouse models of human CRCLM were established to test the anti-metastasis effect of SC and oxaliplatin in the single or combined form. SC exerted an inhibitory effect on the proliferation, invasion, and migration of CRC cells both in vitro and in vivo. The combination index of SC and oxaliplatin was 0.58. In addition, the inhibitory effect of SC in combination with oxaliplatin was found to be significantly stronger compared with that mediated by either SC or oxaliplatin alone, suggesting that SC can effectively enhance the inhibitory effect of oxaliplatin on CRCLM both in vitro and in vivo. SC was also revealed to reverse EMT process in cell lines and tissues, as reflected by the observed downregulation of N-cadherin and vimentin expression and the upregulation of E-cadherin expression. Taken together, data from the present study suggest that SC administration can inhibit CRC cell proliferation, invasion, migration, and EMT, while enhancing the inhibitory effects of oxaliplatin both in vitro and in vivo. These findings indicate (that) SC to be a promising anti-metastasis agent for CRCLM.

Sophocarpine exert protective effect against ox-LDL-induced endothelial damage via regulating NF-κB signaling pathway.

Oxidized low-density lipoprotein (ox-LDL) was known to induce endothelial cell injury to the progression of atherosclerosis (AS). Sophocarpine (SPC), a compound of sophora alkaloids isolated from the plant Sophora alopecuroides, has been shown to exhibit various pharmacological activities. This study was designed to investigate the protective effect of SPC on ox-LDL-induced endothelial cells and explored its underlying mechanism. Our results show that SPC pre-incubation ameliorated ox-LDL-mediated HAECs cytotoxicity, DNA fragmentation, and apoptosis in a dose-dependent manner. Moreover, SPC significantly downregulated the mRNA or protein expression level of pro-inflammatory mediators (TGF-β, IL-6, IL-1β, TNF-α) and pro-inflammatory vascular adhesion molecules (VCAM-1, ICAM-1, and E-selectin). Mechanistically, SPC pre-treatment downregulated IκBα expression and inhibited translocation of NF-κB in ox-LDL-mediated HAECs, overexpression of NF-κB p65 counteracted the cytoprotective and anti-apoptotic effect of SPC, suggesting that its action is dependent on NF-κB signaling pathway. Collectively, SPC suppresses ox-LDL-induced HAECs injury by inhibiting the NF-κB signaling pathway.

Sophocarpine ameliorates cardiac hypertrophy through activation of autophagic responses.

Mounting evidences indicate that autophagy is an essential homeostatic mechanism to maintain the global cardiac structure function. Sophocarpine (SOP), a major bioactive compound derived from the natural plant Sophora flavescens. However, the role of SOP in cardiac hypertrophy remain to be fully elucidated. In the present study, we tested the hypothesis that SOP protects against Ang II-induced cardiac hypertrophy by mediating the regulation of autophagy. The results demonstrated that SOP attenuated the Ang II-induced cardiac hypertrophy, as assessed by measurements of echocardiography parameters, the ratios of heart weight/body weight and left ventricle weight/body weight, histopathological staining, cross-sectional cardiomyocyte area, and the expression levels of cardiac hypertrophic markers. The anti-hypertrophic effect of SOP was mediated by activating autophagy-related pathway, as revealed by reversal of the increased autophagy marker protein expression. These findings reveal a novel mechanism of SOP attenuating cardiac hypertrophy via activating autophagy-related signaling pathways.

Sophocarpine Suppresses NF-κB-Mediated Inflammation Both In Vitro and In Vivo and Inhibits Diabetic Cardiomyopathy.

Diabetic cardiomyopathy (DCM) is a leading cause of mortality in patients with diabetes. DCM is a leading cause of mortality in patients with diabetes. We used both in vitro and in vivo experiments to investigate the hypothesis that sophocarpine (SPC), a natural quinolizidine alkaloid derived from a Chinese herb, could protect against DCM. We used hyperglycemic myocardial cells and a streptozotocin (STZ)-induced type 1 diabetes mellitus mouse model. SPC protected myocardial cells from hyperglycemia-induced injury by improving mitochondrial function, suppressing inflammation, and inhibiting cardiac apoptosis. The SPC treatment significantly inhibited the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling in high-glucose-stimulated inflammatory responses. Moreover, SPC significantly slowed the development and progression of DCM in STZ-induced diabetic mice. These results show that SPC suppresses NF-κB-mediated inflammation both in vitro and in vivo and may be used to treat DCM.

Sophocarpine Attenuates Chronic Constriction Sciatic Nerve Injury-induced Neuropathic Pain in Mice by Inhibiting the HMGB1/TLR4/NF-κB Signaling Pathway

Background: Sophocarpine (SC) is a major alkaloid extracted from Sophora alopecuroides L. Our previous study showed that SC has analgesic effects on neuropathic pain induced by chronic constriction injury (CCI). However, the exact analgesic mechanism of SC on neuropathic pain has not yet been elucidated. Objectives: The current study aimed to examine the anti-neuropathic pain effects of SC on the HMGB1/TLR4/NF-κB signaling pathway to explore its analgesic mechanism in neuropathic pain. Methods: In this experimental study, the neuropathic pain mouse model was established by CCI of the sciatic nerve in a university-affiliated animal lab, China, 2016. Mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), cold withdrawal threshold (CWT), and tail-curling latency (TCL) were used to assess the antinociceptive effect of SC in neuropathic pain mice. The mRNA and protein expression levels of HMGB1, TLR4, NF-κB, p-NF-κB, TNF-α, and IL-6 in the spinal cord of neuropathic pain mice were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Results: Treatment with 40 and 20 mg/kg of SC was effective in increasing MWT, lengthening TWL, reducing CWT, and prolonging TCL in mice with neuropathic pain induced by CCI. Compared to the neuropathic-pain model group, treatment with 40 mg/kg of SC could effectively down-regulate HMGB1, TLR4, NF-κB, p-NF-κB, TNF-α, and IL-6 mRNA and protein expression levels in the spinal cord of mice with neuropathic pain induced by CCI. Conclusions: Our results showed that SC has analgesic effects on neuropathic pain induced by CCI, and its analgesic mechanism may be related to down-regulating the HMGB1/TLR4/NF-κB signaling pathway.

Protective effect of sophocarpine on lipopolysaccharide-induced acute lung injury in mice

Sophocarpine (SOP) is a tetracyclic quinolizidine alkaloid isolated from Sophora alopecuroides L. A number of studies have shown that SOP has anti-inflammatory actions and protects against a variety of tissue and organ injuries. The purpose of this study was to investigate the protective effects of SOP on LPS-induced acute lung injury (ALI) in mice. Lung histological alterations, edema, and MPO activity were measured in this study. Furthermore, the production of inflammatory cytokines and the expression of NF-κB and MAPKs signaling pathways were measured. The results showed that the LPS-induced lung histological alterations, edema, protein concentration, inflammatory cell level in BALF, MDA content, and MPO activity were significantly attenuated by SOP. The LPS-induced inflammatory cytokine TNF-α, IL-1β, and IL-6 were also inhibited by SOP. SOP also inhibited the LPS-induced IL-6 and IL-8 production in A549 cells. Western blot analysis demonstrated that SOP remarkably inhibited the phosphorylation of IκBα and NF-κB. LPS-induced MAPKs activation and TLR4 expression were also suppressed by treatment with SOP. In conclusion, the results indicate that SOP protects against LPS-induced ALI by inhibiting TLR4 signaling pathway.

The protective effect of sophocarpine in osteoarthritis: An in vitro and in vivo study

BackgroundOsteoarthritis (OA) is a type of degenerative joint disease affecting millions of individuals worldwide. However, there are currently no great inflammatory treatments available for it. Sophocarpine (SPC), one of the key bioactive compounds derived from Sophora flavescens, has shown remarkable anti-inflammatory effects. MethodsIn this study, we evaluated the effect of SPC on preventing the progression of OA and investigated its molecular target involved. In brief, rat chondrocytes were pretreated with SPC and subsequently stimulated with IL-1β. We found that SPC reduced the production of pro-inflammatory cytokines, such as nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6). SPC also inhibited the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) at both the gene and protein level. Moreover, SPC promoted the expression of anabolic factors Sox-9 and aggrecan, while inhibiting the expression of catabolic factors, such as matrix metalloproteinases 13 (MMP-13) and thrombospondin motifs 5 (ADAMTS-5) in rat chondrocytes. Mechanistically, we found that SPC inhibited nuclear factor kappa B (NF-κB) via the phosphatidylinositol 3 kinase (PI3K)/AKT pathway. The beneficial effects of SPC were also observed in vivo using a rat OA model. ConclusionsOur findings indicate that SPC may be a potential novel therapeutic in the treatment of OA.

Sophocarpine against enterovirus 71 in vitro.

Sophocarpine (SCA) is a bioactive alkaloid present in Sophoraflavescens Ait. The present study demonstrated that SCA inhibited enterovirus 71 (EV71) infection in Vero cells. The results indicated that the 50% cytotoxicity concentration of SCA for Vero cells was 1,346 µg/ml, and the 50% inhibition concentration of SCA against EV71 was 350 µg/ml. SCA produced a marked inhibitory effect against EV71 when the Vero cells were treated with SCA prior to infection with the virus. Additionally, SCA was effective against EV71 when the Vero cells were infected with EV71 (100xTCID50) that had been treated with SCA for 2 h, and was effective when the Vero cells were infected with EV71 (100xTCID50) at 37°C under 5% CO2 for 2 h prior to treatment with SCA for 2 h. SCA was demonstrated to inhibit the attachment and penetration of EV71 and was more effective at inhibiting attachment. The assay additionally verified that SCA suppressed the replication of viral genomic RNA and indicated that SCA may inhibit EV71 infection in vitro.

Targeting miR-21 with Sophocarpine Inhibits Tumor Progression and Reverses Epithelial-Mesenchymal Transition in Head and Neck Cancer

A major challenge for cancer chemotherapy is the development of safe and clinically effective chemotherapeutic agents. With its low toxicity profile, sophocarpine (SC), a naturally occurring tetracyclic quinolizidine alkaloid derived from Sophora alopecuroides L, has shown promising therapeutic properties, including anti-inflammatory, anti-nociceptive, and antivirus activities. However, the antitumor efficacy of SC and its underlying mechanisms have not been completely delineated. In the present study, the inhibitory effect of SC onhead and neck squamous cell carcinoma (HNSCC) progression and possible mechanisms for this effect involving microRNA-21 (miR-21) regulation were investigated. By cell viability, Transwell, and wound healing assays, we show that SC effectively inhibited proliferation, invasion, and migration of HNSCC cells. Moreover, SC exerted its growth-inhibitory effect via the downregulation of miR-21 expression by blocking Dicer-mediated miR-21 maturation. Furthermore, SC treatment led to the increased expression of PTEN and p38MAPK phosphorylation as well as the reversal of epithelial-mesenchymal transition (EMT), which was rescued by ectopic expression of miR-21 in cells. Notably, SC dramatically repressed tumor growth without observable tissue cytotoxicity in a mouse xenograft model of HNSCC. Our findings offer a preclinical proof of concept for SC as a leading natural agent for HNSCC cancer therapy.

Human microsomal cyttrochrome P450-mediated reduction of oxysophocarpine, an active and highly toxic constituent derived from Sophora flavescens species, and its intestinal absorption and metabolism in rat

Oxysophocarpine (OSC), an active and toxic quinolizidine alkaloid, is highly valued in Sophora flavescens Ait. and Subprostrate sophora Root. OSC is used to treat inflammation and hepatitis for thousands of years in China. This study aims to investigate the CYP450-mediated reduction responsible for metabolizing OSC and to evaluate the absorption and metabolism of OSC in rat in situ. Four metabolites were identified, with sophocarpine (SC) as the major metabolite. SC formation was rapid in human and rat liver microsomes (HLMs and RLMs, respectively). The reduction rates in the liver are two fold higher than in the intestine, both in humans and rats. In HLMs, inhibitors of CYP2C9, 3A4/5, 2D6, and 2B6 had strong inhibitory effects on SC formation. Meanwhile, inhibitors of CYP3A and CYP2D6 had significant inhibition on SC formation in RLMs. Human recombinant CYP3A4/5, 2B6, 2D6, and 2C9 contributed significantly to SC production. The permeability in rat intestine and the excretion rates of metabolites were highest in the duodenum (p<0.05), and the absorbed amount of OSC in duodenum and jejunum was concentration-dependent. The metabolism could be significantly decreased by CYP3A inhibitor ketoconazole. In conclusion, the liver was the main organ responsible for OSC metabolism. First-pass metabolism via CYP3A4/5, 2B6, 2D6, and 2C9 may be the main reason for the poor OSC bioavailability.

Sophocarpine Attenuates the Na+-dependent Ca2+ Overload Induced by Anemonia Sulcata Toxin—Increased Late Sodium Current in Rabbit Ventricular Myocytes

Many studies indicate that an increase in late sodium current (I(Na.L)) of cardiomyocytes causes intracellular Na overload and subsequently raises the reverse Na/Ca exchanger current (INCX), ultimately resulting in intracellular Ca overload. Therefore, using drugs to inhibit the increased INa.L under various pathological conditions can lower intracellular Ca overload. This study was intended to explore the effect of sophocarpine (SOP) on the increase in INa.L, INCX, calcium transient and contraction in rabbit ventricular myocytes induced by Anemonia sulcata toxin II (ATX II), an opener of sodium channel, with the application of whole-cell patch-clamp techniques, the video-based motion edge detection system, and the intracellular calcium concentration determination system. The results indicate that tetrodotoxin (TTX, 4 μM ) obviously decreased INa.L and INCX enlarged by ATX II (30 nM), and SOP (20, 40, and 80 μM) also inhibited both the parameters concentration dependently in rabbit ventricular myocytes. However, transient sodium current remained unaffected by the above-mentioned concentrations of ATX II, TTX, and SOP. In addition, SOP also reversed diastolic calcium concentration, calcium transient amplitude, and ventricular muscle contractility augmented by ATX II. Its effects were similar to those of TTX, a specific inhibitor of the sodium channel. In conclusion, SOP inhibits INa.L, INCX, diastolic Ca concentration, and contractility in rabbit ventricular myocytes, which suggests that relief of intracellular Ca overload through inhibiting INa.L is likely to become a new therapeutic mechanism of SOP against arrhythmia and myocyte damage associated with intracellular Ca overload.

HERG K+ Channel Blockade by the Novel Antiviral Drug Sophocarpine

Human ether-à-go-go-related gene (HERG) encodes the rapid component of the cardiac delayed rectifier K+ current, which has an important role in the repolarization of the cardiac action potential. QT interval prolongation through HERG channel inhibition is associated with a risk of torsade de pointes arrhythmias and is a major challenge for drug development. The effects of the novel antiviral drug sophocarpine (SC) were examined on stably expressed HERG channels in human embryonic kidney (HEK293) cells using a whole-cell patch clamp technique, Western blot analysis and immunofluorescence experiments. SC inhibited HERG channels in a concentration-dependent manner, with an IC50 of 100-300 microM. SC significantly accelerated channel inactivation, recovery from inactivation and onset of inactivation. In addition, it had no effect on channel activation and deactivation. Based on Western blot and immunofluorescence results, SC had no significant effect on the expression of HERG protein. In summary, SC is a potent blocker of HERG K+ channels that functions by changing the channel inactivation kinetics. In addition, SC has no effect on the generation and trafficking of HERG protein.

Simultaneous quantification of oxysophocarpine and its active metabolite sophocarpine in rat plasma by liquid chromatography/mass spectrometry for a pharmacokinetic study

A sensitive, rapid and specific method for the simultaneous quantification of oxysophocarpine (OSC) and its active metabolite sophocarpine (SC) in rat plasma was developed and validated, using a liquid-liquid extraction procedure followed by liquid chromatography/electrospray ionization mass spectrometric (LC/ESI-MS) analysis. The separation was performed on a Zorbax Extend-C(18) column (2.1 mm i.d. x 50 mm, 5 microm) with a C(18) guard column using methanol-water containing 5 mm ammonium acetate (15:85, v/v) as mobile phase. Analysis was performed in selected ion monitoring (SIM) mode with an electrospray ionization (ESI) interface. [M + H](+) at m/z 263 for OSC, [M + H](+) at m/z 247 for SC and [M + H](+) at m/z 249 for matrine (internal standard) were selected as detecting ions, respectively. The method was linear in the concentration ranges 10-1000 ng/mL for OSC and 5-500 ng/mL for SC. The intra- and inter-day precisions (coefficient of variation) were within 7% for both analytes. Their accuracy (relative error) ranged from -6.4 to 1.5%. The limits of detection for OSC and SC were 3 and 1.5 ng/mL, respectively. The limits of quantitation for OSC and SC were 10 and 5 ng/mL, respectively. Recoveries of both analytes were greater than 85% at the low, medium and high concentrations. Both analytes were stable during all sample storage, preparation and analytic procedures. The method was successfully applied to a pharmacokinetic study after an oral administration of OSC to rats with a dose of 15 mg/kg.

Determination of Sophocarpine, Matrine, and Sophoridine in KUHUANG Injection by GC-MS

A GC-MS procedure was developed to determine sophocarpine (SC), matrine (MT), and sophoridine (SRI) in KUHUANG injection. The chromatographic separation was performed on an HP-5MS column (30 m × 0.35 mm inner diameter, 0.25 µm film thickness) with helium as the carrier gas. The oven temperature was programmed from 170 to 205°C at a rate of 0.8 K/min, and n-tetracosane was used as the internal standard (IS). There was a good linear relationship between the peak area ratio of analyte to IS and the concentration of analyte in the ranges 0.0360–0.100 mg/mL for SC, 0.0461–0.346 mg/mL for MT, and 0.0473–0.355 mg/mL for SRI. The recoveries were 86.1–100.9% for SC, 87.9–104.0% for MT, and 92.5–107.2% for SRI. The interday and intraday relative standard deviations (RSDs) of determinations were less than 4.6, 2.7, and 5.1% for the assay of SC, MT, and SRI, respectively. The limits of detection (LOD) were 0.018 mg/mL for SC, 0.0231 mg/mL for MT, and 0.0287 mg/mL for SRI. The limits of quantitation (LOQ) were 0.036 mg/mL for SC, 0.0461 mg/mL for MT, and 0.0473 mg/mL for SRI. The results indicate that the developed method can be used to improve the quality control of KUHUANG injection.