The time course of acute kidney injury and hypokalemia remains unelucidated. We investigated whether altered renal function impacts hypokalemia and clinical predictors for acute kidney injury in patients who used Yokukansan preparation. We performed a secondary analysis of retrospective observational cohort data from adult patients who started Yokukansan preparation. The study was conducted from June 2015 to May 2019 at Tokyo Women’s Medical University, Medical Center East. The effect of acute kidney injury (>1.5-fold increase from baseline serum creatinine level) or renal function recovery on hypokalemia (serum potassium level <3.0 mEq/L) was investigated. The clinical predictors for acute kidney injury were determined using a multivariate Cox proportional hazard analysis. Out of 258 patients, 12 patients had both outcomes, and all but one patient experienced in the order of acute kidney injury and hypokalemia. Excluding one patient, hypokalemia occurred in 11/34 (32%) patients after acute kidney injury and 27/223 (12%) patients without acute kidney injury (p = 0.005). Hypokalemia occurred in 9/25 (36%) of acute kidney injury with recovery, 2/9 (22%) of acute kidney injury without recovery, and 27/223 (12%) of no acute kidney injury (p = 0.014). Patients with acute kidney injury showed a late onset of hypokalemia compared with those without acute kidney injury (p = 0.001). In 258 patients, multivariate Cox proportional hazard analysis showed that high systolic blood pressure and mean arterial pressure increased the risk of acute kidney injury. Clinicians should remember that hypokalemia developed after acute kidney injury while Yokukansan preparation treatment.

Blood transport proteins are biogenic molecules with unique and interesting inherent characteristics that make up living organisms. As the utilization of their inherent characteristics can be a groundbreaking strategy to resolve and improve several clinical problems, attempts have been made to develop pharmaceutical and biomedical preparations based on blood transport proteins for the treatment and diagnosis of disorders. Among various blood transport proteins, we focus on the immense potential of hemoglobin and albumin to serve as carriers of biomedical gases (oxygen and carbon monoxide) and anticancer agents (low-molecular compounds and antisense oligodeoxynucleotides), respectively, for the development of innovative drug delivery systems (DDS) to treat intractable disorders and solid cancers. In this review, I introduce the pharmaceutical technology, strategies, and application of DDS carriers that have been designed on the basis of the structure and function of hemoglobin and albumin. In addition, the prospect of using hemoglobin and albumin as materials for DDS carriers is discussed.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation and structural destruction of the joints. Bone damage occurs in an early stage after onset and osteoclast activation plays a substantial role in its progression. Colony stimulating factor 1 receptor (CSF1R) is a receptor protein tyrosine kinase specifically expressed in monocytic-lineage cells such as macrophages and osteoclasts. Here, we investigated the effect of JTE-952, a novel CSF1R tyrosine kinase inhibitor, on osteoclast formation in vitro and on bone destruction in a mouse model of collagen-induced arthritis. JTE-952 completely inhibited osteoclast differentiation from human monocytes, with an IC₅₀ of 2.8 nmol/L, and reduced osteoclast formation from the synovial cells of RA patients. Detectable levels of colony stimulating factor 1 (CSF1), a ligand of CSF1R, were observed in the synovial tissues of the arthritis model, similar to those observed in the pathology of human RA. JTE-952 significantly suppressed increases in the bone destruction score, the number of tartrate-resistant-acid-phosphatase-positive cells, and the severity of arthritis in the model mice. We also examined the efficacy of JTE-952 combined with methotrexate. This combination therapy more effectively reduced the severity of bone destruction and arthritis than monotherapy with either agent alone. In summary, JTE-952 potently inhibited human osteoclast formation in vitro and suppressed bone destruction in an experimental arthritis model, especially when combined with methotrexate. These results indicate that JTE-952 should strongly inhibit bone destruction and joint inflammation in RA patients and effectively prevent the progression of the structural destruction of joints.

Signal transducer and activator of transcription 3 (STAT3) is considered a potential target for cancer treatment because of its relationship with cellular transformation and tumor initiation and progression. In this study, we aimed to identify a new anti-cancer drug candidate from natural products by targeting STAT3 activity. Using STAT3-luciferase reporter cell line, we screened the chemical library of natural products and found that baicalein, a flavone isolated from the roots of Scutelleria baicalensis, strongly suppressed STAT3 activity in breast cancer cells. Baicalein inhibited STAT3 transcriptional activity and its phosphorylation, and further exhibited anti-proliferative effects in breast cancer cells. Moreover, baicalein suppressed the production of interleukin (IL)-6 and the metastatic potential of breast cancer cells both in vitro and in vivo. Collectively, our study suggests baicalein as an attractive phytochemical compound for reducing metastatic potential of breast cancer cells by regulating STAT3 activity.

Royal jelly (RJ) is known as an important functional foodstuff that promotes several health benefits and contains various bioactive substances, including major royal jelly proteins (MRJPs). Among the MRJPs, MRJP3 possesses both cell proliferation and wound healing effects. As the carboxyl domain of MRJP3 contains tandem penta-peptide repeat (TPR) sequences unique to MRJP3 among the MRJPs, we purified the TPRs as glutathione-S-transferase (GST)-fusion proteins and demonstrated their dose-dependent effects on THP-1 and Vero cell proliferation. The GST-TPR protein with 19 repeats (GST-TPR19) showed cell proliferative activity equivalent to MRJP3 and higher than GST-TPR6. GST-TPR19 also exhibited wound healing activity at a level similar to MRJP3. Digestion of GST-TPR19 with trypsin had no effect on its cell proliferative activity, suggesting that the main digested products; i.e., penta-peptides (Q-N-x-N-[K/R]), maintain the cell proliferative ability of MRJP3. In conclusion, the TPRs of MRJP3 are critical to the beneficial effect(s) of RJ.

Nanoparticles are used in many fields and in everyday products. Silver nanoparticles are the most frequently used nanoparticles; for example, in food-related products, owing to their antibacterial activity. However, it has been pointed out that they might have unexpected biological effects, and evaluation of their effects is underway. Although there is a growing body of evidence that nanoparticles can also induce epigenetic changes, there is still little information on the underlying mechanisms. Here, we evaluated changes in DNA methylation induced by silver nanoparticles and attempted to elucidate the induction mechanism. Immunofluorescence staining analysis revealed that silver nanoparticles with a diameter of 10, 50, or 100 nm (nAg10, nAg50, and nAg100, respectively) decreased the content of methylated DNA in A549 alveolar epithelial cells. The level of DNA methyltransferase 1 (Dnmt1) protein, which is involved in maintaining methylation during DNA replication, was significantly decreased, whereas that of Dnmt3b, which is responsible for de novo DNA methylation, was significantly increased by nAg10 treatment. Co-treatment with nAg10 and cycloheximide, which inhibits translation by inhibiting the translocation step of protein synthesis, decreased the level of Dnmt1 in comparison with nAg10-treated A549 cells, indicating a post-translational effect of nAg10. Furthermore, pretreatment with the proteasome inhibitor lactacystin restored the levels of Dnmt1 protein and DNA methylation in nAg10-treated cells. Collectively, these results suggest that nAg10 induced DNA hypomethylation through a proteasome-mediated degradation of Dnmt1.

Gene and nucleic acid-based medication is an ultimate strategy in the field of personalized medicine. A gene or short interference RNA (siRNA) molecule needs to be delivered to the appropriate organelle (i.e., nucleus and cytoplasm, respectively). We recently focused on improving the intrinsic activity of my original material (ssPalm) in terms of endosomal/lysosomal membrane destabilization activity by chemically modifying the tertiary amine structure. In parallel, I have been expanding the range of applications of ssPalms. The first application is a DNA or RNA vaccine. My crucial finding is that the vitamin E-scaffold ssPalm (ssPalmE) is highly immune-stimulative when combined with DNA. Thereafter, I redesigned the hydrophobic scaffold structure, and found that an oleic acid-scaffold ssPalm (ssPalmO) can confer anti-inflammatory characteristics. Based on this result, I further upgraded the ssPalmO, by inserting a newly designed linker with self-degradable properties.

Matrix metalloproteinases (MMPs) are required for collagen degradation which play a key pathological role in arthritis progression. Herein, the effect of sinomenine (SN) on Interleukin 1 beta (IL-1β)-induced MMPs production and its underlying mechanism were explored in SW1353 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that 200 and 400 µM SN significantly inhibited SW1353 cell proliferation, thus the lower dose of SN (25–100 µM) were used in the subsequent experiments. Notably, the increased mRNA and protein levels of suppressor of cytokine signaling (SOCS) 3 were dose-dependently induced by SN. SN significantly suppressed mRNA and protein levels of MMPs in IL-1β-induced SW1353 cells. Through Western blot analysis, SN showed inhibitory effect on IL-1β-induced TAK1 and p65 phosphorylation. Moreover, SN blocked the interaction of TRAF6 and TAK1 resulting in inactivation of IL-1β pathway. Mechanistically, the inhibitory effect of SN on MMPs levels alongside TRAF6 and TAK1 interactions was abrogated by silencing SOCS3. Moreover, SN did not inhibit TAK1 kinase activity. In TAK1 silencing cells, the levels of MMPs and p65 phosphorylation of SN-treatedcells were lower than dimethyl sulfoxide (DMSO)-treated cells, indicating that blocking interaction was not a unique way for SN to inhibit MMPs levels. Finally, SN significantly inhibited IL-6-induced Janus tyrosine kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) phosphorylation in SW1353 cells. The levels of JAK2 phosphorylation and MMPs did not show a significant difference between IL-6 + SOCS3-small interfering RNA (siRNA) + SN group and IL-6 + SOCS3-siRNA + DMSO group. These findings demonstrated that SOCS3 expression was increased by SN blocked IL-1β-induced interaction between TRAF6 and TAK1 as well as IL-6 pathway activation, thereby culminating in the inhibition of MMPs levels.

Cisplatin is a widely used chemotherapy for solid tumors; however, its benefits are limited by serious nephrotoxicity, particularly in proximal tubular cells. The present study investigated the renoprotective effect and mechanisms of germacrone, a bioactive terpenoid compound found in Curcuma species on cisplatin-induced toxicity of renal cells. Germacrone (50 and 100 µM) attenuated apoptosis of human renal proximal tubular cells, RPTEC/TERT1 following treatment with 50 µM cisplatin and for 48 h. Co-treating RPTEC/TERT1 cells with cisplatin and germacrone significantly reduced cellular platinum content compared with cisplatin treatment alone. The effect of germacrone on organic cation transporter 2 (OCT2) which is a transporter responsible for cisplatin uptake was determined. Germacrone showed an inhibitory effect on OCT2-mediated methyl-4-phenylpyridinium acetate (³H-MPP⁺) uptake with IC₅₀ of 15 µM with less effect on OCT1. The germacrone’s protective effect on cisplatin-induced cytotoxicity was not observed in cancer cells; cisplatin’s anti-cancer activity was preserved. In conclusion, germacrone prevents cisplatin-induced toxicity in renal proximal tubular cells via inhibition OCT2 transport function and reducing cisplatin accumulation. Thus germacrone may be a good candidate agent used for reducing cisplatin-induced nephrotoxicity.

Lubiprostone is an effective drug for various types of constipation in patients without cancer; however, there is no report on its efficacy and safety in patients with cancer. Our purpose was to evaluate the efficacy and safety of lubiprostone for constipation in cancer patients. We retrospectively studied 124 patients (cancer, N = 67) who were treated with lubiprostone for constipation in our hospital between June 2013 and May 2016. The number of bowel movements (BMs) increased in the both the cancer and non-cancer groups. The mean change in BM frequency did not differ between the two groups. Approximately 70% of patients in both groups had an initial BM within 24 h after administration of lubiprostone. The most common lubiprostone-related adverse events in both groups were diarrhea (38.8 vs. 14%), and nausea (22.4 vs. 8.8%). No lubiprostone-related serious adverse events occurred. Discontinuation due to the side effects of lubiprostone was more frequent in cancer patients (p = 0.023). Logistic regression analysis showed that the risk of discontinuation of lubiprostone in cancer patients was high in patients with a body-mass index (BMI) <22, and low in patients using opioids and magnesium oxide dosage ≥1000 mg/d. Our study showed that while lubiprostone was as effective in cancer patients as in non-cancer patients, in cancer patients it was associated with a high incidence of diarrhea and nausea side effects and warranted caution, especially in patients with a low BMI.

Xanthine and hypoxanthine are intermediate metabolites of uric acid and a source of reactive oxidative species (ROS) by xanthine oxidoreductase (XOR), suggesting that facilitating their elimination is beneficial. Since they are reabsorbed in renal proximal tubules, we investigated their reabsorption mechanism by focusing on the renal uric acid transporters URAT1 and GLUT9, and examined the effect of clinically used URAT1 inhibitor on their renal clearance when their plasma concentration is increased by XOR inhibitor. Uptake study for [³H]xanthine and [³H]hypoxanthine was performed using URAT1- and GLUT9-expressing Xenopus oocytes. Transcellular transport study for [³H]xanthine was carried out using Madin–Darby canine kidney (MDCK)II cells co-expressing URAT1 and GLUT9. In in vivo pharmacokinetic study, renal clearance of xanthine was estimated based on plasma concentration and urinary recovery. Uptake by URAT1- and GLUT9-expressing oocytes demonstrated that xanthine is a substrate of URAT1 and GLUT9, while hypoxanthine is not. Transcellular transport of xanthine in MDCKII cells co-expressing URAT1 and GLUT9 was significantly higher than those in mock cells and cells expressing URAT1 or GLUT9 alone. Furthermore, dotinurad, a URAT1 inhibitor, increased renal clearance of xanthine in rats treated with topiroxostat to inhibit XOR. It was suggested that xanthine is reabsorbed in the same manner as uric acid through URAT1 and GLUT9, while hypoxanthine is not. Accordingly, it is expected that treatment with XOR and URAT1 inhibitors will effectively decrease purine pools in the body and prevent cell injury due to ROS generated during XOR-mediated reactions.

The usefulness of the urine protein : creatine ratio (UPCR) in management of molecular targeted therapy and immunotherapy has not been studied, although urine protein dipstick testing (uPr) is widely used in the clinical setting. The aim of this study was to investigate the usefulness of UPCR as compared to uPr in patients undergoing molecular targeted therapy for advanced renal cell carcinoma (RCC). A total of 25 patients (median age 68 years) with advanced RCC were included. Sunitinib, pazopanib, axitinib, sorefenib, everolimus, and nivolumab were administered to 15, 9, 16, 3, 7, and 13 patients, respectively, with duplication. Proteinuria was managed according to the grade determined by UPCR. Data at every treatment visit were retrospectively collected and uPr and UPCR were compared. The overall incidences of any grade of proteinuria associated with sunitinib, pazopanib, axitinib, sorafenib and everolimus were 86.7, 88.9, 93.8, 100, and 85.7%, respectively. There were discordances between the uPr-based grade and UPCR-based grade. UPCR did not meet the criteria of Grade 3 in 70.6, 100, 83.3, and 83.3% at visits in cases with uPr 3+ for sunitinib, pazopanib, sorafenib, and everolimus, respectively. In axitinib treatment, UPCR did not meet the criteria for withholding in 46.2% of the cases of uPr 2+ and more. Our study suggests that UPCR may be useful tool in management of adverse events associated with tyrosine kinase inhibitors, everolimus and can provide patients with optimal opportunities for receiving treatment.

Sinomenine was found to play anti-cancer functions in different type of cancers, while the mechanisms underlying the anticancer effects of sinomenine in retinoblastoma (RB) remains unclear. The present study was designed to explore the impacts of sinomenine on cell proliferation and invasion ability of RB cells and the related mechanism. Human retinoblastoma cell line WERI-RB-1 and Y79 cells were cultured and treated by different concentration of sinomenine, and then the proliferation ability of the cells was determined via performing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assay. The cell apoptosis was examined via performing the flow cytometry assay. Then scratch wound healing analysis as well as and transwell invasion analysis have been performed to determine the effect of sinomenine on cell migration ability as well as invasion ability. The proteins level of phosphatidylinositol 3-kinase (PI3K)/AKT signaling molecules were determined with Western blot assay. We found that sinomenine was able to decrease the proliferation and promote the apoptosis of RB cells in a dose-dependent manner; moreover, sinomenine also significantly suppressed the migration as well as invasion ability of WERI-RB-1 and Y79 cells in vitro. Furthermore, sinomenine also de-activated PI3K/AKT signaling in WERI-RB-1 cells via inhibited the phosphorylation of PI3K and AKT proteins. Sinomenine can exert anti-tumor function on RB cells in vitro, therefore sinomenine might be a potential alterative medication for the treatment for RB.

Renal insufficiency secondary to contrast administration remains a prevalent and debilitating complication of angiographic procedures. Contrast-induced nephropathy (CIN) is a common clinical problem for which there is no effective medical treatment. However, agmatine has been shown to be effective against ischemia/reperfusion-induced acute kidney injury in rats, a similar condition to CIN. Our aim was to examine the protective effects of agmatine in a rat model of CIN and, based on those results, in a rabbit model of CIN. CIN in the rat model was induced by intravenous administration of indomethacin (10 mg/kg), N^ω-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg) and iopamidol (OYPALOMIN, 7.4 g iodine/kg) at 2 weeks after a unilateral nephrectomy. CIN in the rabbit model was induced by intrarenal arterial injection of only iopamidol (BYSTAGE, 4.8 g iodine/kg). Intravenous injection of agmatine (0.1 and 0.3 mmol/kg) did not attenuate the CIN-induced renal insufficiency in the rat model. Intravenous injection of agmatine (0.3 mmol/kg) attenuated the CIN-induced renal insufficiency in the rabbit model such as increases in blood urea nitrogen and plasma creatinine levels. Renal histological damage was also improved by the agmatine administration. The difference in effects of agmatine injection between CIN rats and CIN rabbits was caused by indomethacin and L-NAME administrations. These results indicate that agmatine prevents the development of CIN-induced renal insufficiency in rabbits, and the effect is accompanied by activation of nitric oxide synthase and subsequent increase of blood flow.

trans-Fatty acids (TFAs) are unsaturated fatty acids with at least one carbon–carbon double bond in trans configuration. TFA consumption has been epidemiologically associated with neurodegenerative diseases (NDs) including Alzheimer’s disease. However, the underlying mechanisms of TFA-related NDs remain unknown. Here, we show a novel microglial signaling pathway that induces inflammation and cell death, which is dramatically enhanced by elaidic acid (EA), the most abundant TFA derived from food. We found that extracellular ATP, one of the damage-associated molecular patterns (DAMPs) leaked from injured cells, induced activation of the apoptosis signal-regulating kinase 1 (ASK1)-p38 pathway, which is one of the major stress-responsive mitogen-activated protein (MAP) kinase signaling pathways, and subsequent caspase-3 cleavage and DNA ladder formation (hallmarks of apoptosis) in mouse microglial cell lines including BV2 and MG6 cells. Furthermore, we found that in these microglial cell lines, EA, but not its cis isomer oleic acid, facilitated extracellular ATP-induced ASK1/p38 activation and apoptosis, which was suppressed by pharmacological inhibition of either p38, reactive oxygen species (ROS) generation, P2X purinoceptor 7 (P2X₇), or Ca²⁺/calmodulin-dependent kinase II (CaMKII). These results demonstrate that in microglial cells, extracellular ATP induces activation of the ASK1-p38 MAP kinase pathway and ultimately apoptosis downstream of P2X₇ receptor and ROS generation, and that EA promotes ATP-induced apoptosis through CaMKII-dependent hyperactivation of the ASK1-p38 pathway, in the same manner as in macrophages. Our study may provide an insight into the pathogenesis of NDs associated with TFAs.

Chronic kidney disease (CKD) patients with secondary hyperparathyroidism (SHPT) have an increased risk of cardiovascular disease (CVD). Cinacalcet is a calcimimetic that permits impaired endothelial functions to be recovered via inhibiting parathyroid hormone (PTH) production in SHPT patients. However, the underlying mechanism for its action remains unknown. The purpose of this study was to examine the effect of cinacalcet on the redox state of human serum albumin (HSA), a reliable marker for assessing endothelial oxidative damage in SHPT patients who were receiving hemodialysis. Cinacalcet was administered to six SHPT patients for a period of 8 weeks. After 4 weeks of treatment, cinacalcet significantly decreased the oxidized albumin ratio which is a ratio of reduced and oxidized forms of HSA via increasing reduced form of HSA. Moreover, the radical scavenging abilities of HSA that was isolated from SHPT patients were increased by cinacalcet, suggesting the recovery of the impaired vascular anti-oxidant ability. Interestingly, the oxidized albumin ratio in SHPT patients was significantly higher than that in hemodialysis patients. In addition, the changes of intact PTH levels were significantly correlated with the oxidized albumin ratio. It therefore appears that PTH may induce oxidative stress in SHPT patients. In fact, an active analogue of PTH increased the production of reactive oxygen species in human endothelial cells. Thus, cinacalcet exhibits anti-oxidative activity through its pharmacological action. Additionally, cinacalcet itself showed radical scavenging activity. In conclusion, cinacalcet improves the redox status of HSA by inhibiting PTH production and partially by its radical scavenging action.

It has long been suggested that a Na⁺-dependent carrier-mediated transport system is involved in the absorption of nucleobases and analogs, including some drugs currently in therapeutic use, for their uptake at the brush border membrane of epithelial cells in the small intestine, mainly based on studies in non-primate experimental animals. The presence of this transport system was indeed proved by the recent identification of sodium-dependent nucleobase transporter 1 (SNBT1/Slc23a4) as its molecular entity in rats. However, this transporter has been found to be genetically deficient in humans and higher primates. Aware of this deficiency, we need to revisit the issue of the absorption of these compounds in the human small intestine so that we can understand the mechanisms and gain information to assure the more rational use and development of drugs analogous to nucleobases. Here, we review the current understanding of the intestinal absorption of nucleobases and analogs. This includes recent knowledge about the efflux transport of those compounds across the basolateral membrane when exiting epithelial cells, following brush border uptake, in order to complete the overall absorption process; the facilitative transporters of equilibrative nucleoside transporter 1 (ENT1/SLC29A1) and equilibrative nucleobase transporter 1 (ENBT1/SLC43A3) may be involved in that in many animal species, including human and rat, without any major species differences.

Vascular dementia (VD) is a common neurodegenerative disease, and the cognitive dysfunction is a major manifestation of VD. Lots of evidences showed that beta-amyloid (Aβ) deposition and neuroinflammation act as vital elements in the progress of VD. The previous studies showed that osthole (OST) can improve the cognitive function of VD and Alzheimer’s disease (AD). However, the effect of OST on Aβ in VD brain is still unclear. Chronic cerebral hypoperfusion (CCH) of rats were used to investigate the effect of OST on Aβ through nod-like receptor protein 3 (NLRP3) inflammasome in this study. Morris Water Maze and Y-maze were used to test the spatial learning, memory and working abilities. Hematoxylin–eosin (H&E) and Nissl staining were used to observe the morphology and number of hippocampal neurons. Immunofluorescence staining was used to observe the number of microglia activated. Western blot was used to detect the expression of proteins. The study results showed that OST obviously enhanced the spatial learning, memory and working abilities induced by modified bilateral common carotid artery occlusion (BCCAO) in rats, improved the pathological damage of hippocampal neurons induced by BCCAO in rats, inhibited the activation of microglia induced by BCCAO in rats. Furthermore, this study also discovered that OST reduced Aβ deposition in VD hippocampus via inhibition the NLRP3 inflammasome. Together, these results suggest that OST reduces Aβ deposition via inhibition NLRP3 inflammasome in microglial in VD.

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium, and it is found in many foods. Acrylamide (AA) can be produced in foods treated at high temperatures. In this study, we investigated the combined toxicity of OTA and AA against human renal and hepatic cells in vitro. The concentration at which the synergistic effect of OTA and AA occurs was determined using the combination index obtained from the cell viability results for OTA and AA individually or in combination. The synergistic toxicity of both substances was evaluated by cell viability and the production of reactive oxygen species. In addition, apoptosis-related markers were significantly upregulated by OTA and AA individually or in combination. To determine the combined toxic effects of OTA and AA on the cells, the levels of enzymes involved in the phase I reaction and apoptosis-related markers were determined using quantitative (q)PCR and Western blot. The expression levels of CYP enzymes CYP1A1 and CYP1A2 involved in the phase I reaction significantly increased when the cells were treated with OTA and AA in combination. The expression of apoptosis-related markers, Bcl2-associated X protein (Bax) and caspase 3, also increased when the cells were treated with OTA and AA in combination. Therefore, the synergistic toxicity of OTA and AA suggests that such effects may contribute to nephrotoxicity and hepatotoxicity.

Crocetin is a major bioactive ingredient in saffron (Crocus sativus L.) and has favorable cardiovascular effects. Here, the effects of crocetin on L-type Ca²⁺ current (I_Ca-L), contractility, and the Ca²⁺ transients of rat cardiomyocytes, were investigated via patch-clamp technique and the Ion Optix system. A 600 µg/mL dose of crocetin decreased I_Ca-L 31.50 ± 2.53% in normal myocytes and 35.56 ± 2.42% in ischemic myocytes, respectively. The current voltage nexus of the calcium current, the reversal of the calcium current, and the activation/deactivation of the calcium current was not changed. At 600 µg/mL, crocetin abated cell shortening by 28.6 ± 2.31%, with a decrease in the time to 50% of the peak and a decrease in the time to 50% of the baseline. At 600 µg/mL, crocetin abated the crest value of the ephemeral Ca²⁺ by 31.87 ± 2.57%. The time to half maximal of Ca²⁺ peak and the time constant of decay of Ca²⁺ transient were both reduced. Our results suggest that crocetin inhibits L-type Ca²⁺ channels, causing decreased intracellular Ca²⁺ concentration and contractility in adult rat ventricular myocytes. These findings reveal crocetin's potential use as a calcium channel antagonist for the treatment of cardiovascular disease.