A Novel Fluorescence-Based Method to Evaluate Ileal Apical Sodium-Dependent Bile Acid Transporter ASBT

This study aimed to demonstrate usefulness of the fluorophore-labeled bile acid derivative, N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2′-aminoethane sulfonate (tauro-nor-THCA-24-DBD) as a substrate of apical sodium-dependent bile acid transporter (ASBT, SLC10A2), which is expressed at distal ileum for reabsorption of bile acids and to find a novel fluorescence-based method to evaluate ASBT activity. In HPLC analysis, chromatogram of tauro-nor-THCA-24-DBD showed double peaks: R- and S-isomers of the compound. When ASBT was expressed in Xenopus laevis oocytes, their uptakes were higher than those by control oocytes, demonstrating both are transported by ASBT. Therefore, results were analyzed separately as peak 1, peak 2 and sum of them. Concentration dependent uptake of tauro-nor-THCA-24-DBD in ASBT-expressing oocytes was saturable with Km 122 μM and Vmax 1.49 pmol/oocyte/30 min for peak 1, 30.7 μM and 1.34 pmol/oocyte/30 min for peak 2, and 40.6 μM and 2.36 pmol/oocyte/30 min for sum, respectively. These uptakes were decreased in the presence of taurocholic acid and in the Na⁺ free condition. Furthermore, in Caco-2 cells, tauro-nor-THCA-24-DBD uptake was also Na⁺-dependent and saturable. Additionally, these uptakes were decreased by elobixibat, a selective ASBT inhibitor. Accordingly, it was concluded that tauro-nor-THCA-24-DBD is a substrate of ASBT and useful to evaluate the intestinal ASBT transport activity.     

Rat Kidney Slices for Evaluation of Apical Membrane Transporters in Proximal Tubular Cells

Kidney slice has been often used as a tool reflecting basolateral transport in renal tubular epithelial cells. Recently, we reported that several important apical reabsorptive transporters such as Octn1/2, Sglt1/2, and Pept1/2 were functional in mouse kidney slices as well as transporter activities in basolateral side, which have been well accepted. Because rats are often used for preclinical pharmacodynamic and pharmacokinetic studies as well as mice, it is important to confirm applicability of rat kidney slices for evaluation of apically expressed transporters. The present study investigates usefulness of kidney slices from rats for evaluation of apical membrane transporters for efflux (multidrug resistance 1a, mdr1a) as well as influx (Octn1/2, Sglt1/2, Pept1/2). Na⁺-dependent uptake of ergothioneine (Octn1), carnitine (Octn2), and methyl-α-D-glucopyranoside (Sglt1/2) by rat kidney slices was observed, and the uptake was decreased by selective inhibitors. In addition, uptake of glycyl-sarcosine (Pept1/2) showed H⁺-dependence and was decreased by selective inhibitor. Furthermore, accumulation of mdr1a substrate azasetron was increased in the presence of zosuquidar, an mdr1a inhibitor, while strain differences existed. In conclusion, rat kidney slices should be useful for evaluation of renal drug disposition regulated by transporters in apical as well as basolateral membranes of rat renal proximal tubule cells.     

Phenolsulfonphthalein as a surrogate substrate to assess altered function of the prostaglandin transporter SLCO2A1

The prostaglandin (PG) transporter SLCO2A1 regulates PGE₂ signaling and interacts with many drugs, and SLCO2A1 defects is associated with PG metabolic disorders. This study aimed to characterize a non-metabolic phenolsulfonphthalein (PSP) transport mediated by SLCO2A1. PSP uptake by HEK293 cells expressing human SLCO2A1 (HEK/2A1 cells) was pH-independent and saturable with a K_m value of 54.5 ± 9.5 μM PGE₂ competitively inhibited PSP uptake with a K_i of 257.3 ± 22.8 nM. When PSP was intravenously (i.v.) injected, concentration-time curve showed a biphasic response. In Slco2a1-deficient (−/−) mice, AUC_inf tented to decrease and the central distribution volume (V₁) significantly increased, compared to wild-type (wt) counterparts. Intriguingly, Slco2a1-deficiency significantly reduced a ratio of tissue-to-plasma concentration in the lungs at 15 min after i.v. injection, suggesting that SLCO2A1 limits tissue distribution of PSP. In conclusion, these results prove that PSP is a potential surrogate for monitoring SLCO2A1 function, providing a new concept for diagnostics for the genetic diseases caused by defects in SLCO2A1 gene.     

Amlodipine alleviates cisplatin-induced nephrotoxicity in rats through gamma-glutamyl transpeptidase (GGT) enzyme inhibition,associated with regulation of Nrf2/HO-1, MAPK/NF-κB,and Bax/Bcl-2 signaling

BackgroundThe therapeutic utility of the effective chemotherapeutic agent cisplatin is hampered by its nephrotoxic effect. We aimed from the current study to examine the possible protective effects of amlodipine through gamma-glutamyl transpeptidase (GGT) enzyme inhibition against cisplatin nephrotoxicity.MethodsAmlodipine (5 mg/kg, po) was administered to rats for 14 successive days. On the 10^th day, nephrotoxicity was induced by a single dose of cisplatin (6.5 mg/kg, ip). On the last day, blood samples were collected for estimation of kidney function, while kidney samples were used for determination of GGT activity, oxidative stress, inflammatory, and apoptotic markers, along with histopathological evaluation.ResultsAmlodipine alleviated renal injury that was manifested by significantly diminished serum creatinine and blood urea nitrogen levels, compared to cisplatin group. Amlodipine inhibited GGT enzyme, which participates in the metabolism of extracellular glutathione (GSH) and platinum-GSH-conjugates to a reactive toxic thiol. Besides, amlodipine diminished mRNA expression of NADPH oxidase in the kidney, while enhanced the anti-oxidant defense by activating Nrf2/HO-1 signaling. Additionally, it showed marked anti-inflammatory response by reducing expressions of p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-kappa B (NF-κB), with subsequent down-regulation of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and vascular cell adhesion molecule-1 (VCAM-1). Moreover, amlodipine reduced Bax/Bcl-2 ratio and elevated hepatocyte growth factor (HGF), thus favoring renal cell survival.ConclusionsEffective GGT inhibition by amlodipine associated with enhancement of anti-oxidant defense and suppression of inflammatory signaling and apoptosis support our suggestion that amlodipine could replace toxic GGT inhibitors in protection against cisplatin nephrotoxicity.     

Selective inhibition of interleukin-1 receptor-associated kinase 1 ameliorates lipopolysaccharide-induced sepsis in mice

Interleukin-1 receptor-associated kinases (IRAKs), particularly IRAK1 and IRAK4, are important in transducing signal from Toll-like receptor 4. We interrogated if a selective inhibition of IRAK1 could alleviate lipopolysaccharide (LPS)-induced sepsis. In this study, we tested the impact of a novel selective IRAK1 inhibitor Jh-X-119-01 on LPS-induced sepsis in mice. Survival at day 5 was 13.3% in control group where septic mice were treated by vehicle, while the values were 37.5% (p = 0.046, vs. control) and 56.3% (p = 0.003, vs. control) for 5 mg/kg and 10 mg/kg Jh-X-119-01-treated mice. Jh-X-119-01 alleviated lung injury and reduced production of TNFα and IFNγ in peritoneal macrophages. Jh-X-119-01 decreased phosphorylation of NF-κB and mRNA levels of IL-6 and TNFα in LPS-treated macrophages in vitro. Jh-X-119-01 selectively inhibited IRAK1 phosphorylation comparing with a non-selective IRAK1/4 inhibitor which simultaneously inhibited phosphorylation of IRAK1 and IRAK4. Both Jh-X-119-01 and IRAK1/4 inhibitor increased survival of septic mice, but Jh-X-119-01-treated mice had higher blood CD11b⁺ cell counts than IRAK1/4 inhibitor-treated ones [24 h: (1.18 ± 0.26) × 10⁶/ml vs. (0.79 ± 0.20) × 10⁶/ml, p = 0.001; 48 h: (1.00 ± 0.30) × 10⁶/ml vs. (0.67 ± 0.23) × 10⁶/ml, p = 0.042]. IRAK1/4 inhibitor induced more apoptosis of macrophages than Jh-X-119-01 did in vitro. IRAK1/4 inhibitor decreased protein levels of anti-apoptotic BCL-2 and MCL-1 in RAW 264.7 and THP-1 cells, an effect not seen in Jh-X-119-01-treated cells. In conclusion, Jh-X-119-01 selectively inhibited activation of IRAK1 and protected mice from LPS-induced sepsis. Jh-X-119-01 showed less toxicity on macrophages comparing with a non-selective IRAK1/4 inhibitor.     

The co-expression characteristics of LAG3 and PD-1 on the T cells of patients with breast cancer reveal a new therapeutic strategy

Many studies have shown a special interaction between LAG3 and PD-1 in T cell inhibition, while the co-expression and effect of LAG3 and PD-1 on T cells in breast cancer patients are still not very clear. Here, with strict exclusion criteria, 88 patients with breast cancer and 18 healthy controls were enrolled. The percentages of LAG3⁺PD-1⁺ T cells in their peripheral blood (PBL) and tumor infiltrating T cells (TIL) were analyzed by flow cytometry, which showed an increase in TILs but no difference in PBLs and presented differences in TILs in different molecular subtypes (P < 0.05). In triple-negative breast cancer (TNBC), the highest percentages were observed, while in ER+/PR+ breast cancer, the lowest percentages were observed; however, these percentages were not different in different clinical stages (P > 0.05). Immunohistochemical staining showed that the expression of their ligands, PD-L1, MHC class II molecular and FGL1, was inconsistent in different molecular subtypes and clinical stages. Analysis of the functions of T cells with different phenotypes showed that the proliferation and secretion capacity of LAG3⁺PD-1⁺ T cells was obviously exhausted, with more than a two-fold of decrease compared with the groups of single positive LAG3 or PD-1 (P < 0.05). Finally, in a mouse model of TNBC, the dual blockade of LAG3 and PD-1 was indicated to achieve a better anti-tumour effect than either one alone (P < 0.05), which may provide a new strategy for the immunoregulatory treatment of patients with TNBC in the future.     

Altered cisplatin pharmacokinetics during nonalcoholic steatohepatitis contributes to reduced nephrotoxicity

Disease-mediated alterations to drug disposition constitute a significant source of adverse drug reactions. Cisplatin (CDDP) elicits nephrotoxicity due to exposure in proximal tubule cells during renal secretion. Alterations to renal drug transporter expression have been discovered during nonalcoholic steatohepatitis (NASH), however, associated changes to substrate toxicity is unknown. To test this, a methionine- and choline-deficient diet-induced rat model was used to evaluate NASH-associated changes to CDDP pharmacokinetics, transporter expression, and toxicity. NASH rats administered CDDP (6 mg/kg, i.p.) displayed 20% less nephrotoxicity than healthy rats. Likewise, CDDP renal clearance decreased in NASH rats from 7.39 to 3.83 mL/min, renal secretion decreased from 6.23 to 2.80 mL/min, and renal CDDP accumulation decreased by 15%, relative to healthy rats. Renal copper transporter-1 expression decreased, and organic cation transporter-2 and ATPase copper transporting protein-7b increased slightly, reducing CDDP secretion. Hepatic CDDP accumulation increased 250% in NASH rats relative to healthy rats. Hepatic organic cation transporter-1 induction and multidrug and toxin extrusion protein-1 and multidrug resistance-associated protein-4 reduction may contribute to hepatic CDDP sequestration in NASH rats, although no drug-related toxicity was observed. These data provide a link between NASH-induced hepatic and renal transporter expression changes and CDDP renal clearance, which may alter nephrotoxicity.     

pH-dependent transport kinetics of the human organic anion-transporting polypeptide 1A2

Organic anion-transporting polypeptide (OATP) 1A2 is expressed on the apical sides of intestinal and renal epithelial cells and considered to be involved in the intestinal absorption and renal reabsorption of drugs. Although the transport activity of OATP1A2 is considered to be pH-dependent, the effects of pH on its kinetic parameters and on the potency of OATP1A2 inhibitors are yet to be elucidated. Some OATP are known to have multiple binding sites (MBS), but it remains unclear whether OATP1A2 has MBS. In the present study, we evaluated the influence of pH on the OATP1A2-mediated uptake of estrone 3-sulfate using OATP1A2-expressing HEK293 cells. The uptake of 0.3 μM estrone 3-sulfate by HEK293-OATP1A2 cells was pH-dependent. OATP1A2 exhibited bimodal saturation kinetics at pH 6.3 and 7.4. Compared with that seen at pH 6.3 (5.62 μM), the K_m value of the high-affinity site was 8-fold higher at pH 7.4 (43.2 μM). In addition, the influence of pH on the potency of inhibitors varied among the examined inhibitors. These results suggest that the transport properties of OATP1A2 under lower pH conditions, such as those found in the microenvironments of the small intestinal mucosa and distal tubules, differ from those seen under neutral pH conditions.     

Cilastatin protects against imipenem-induced nephrotoxicity via inhibition of renal organic anion transporters (OATs)

Imipenem is a carbapenem antibiotic. However, Imipenem could not be marketed owing to its instability and nephrotoxicity until cilastatin, an inhibitor of renal dehydropeptidase-I (DHP-I), was developed. In present study, the potential roles of renal organic anion transporters (OATs) in alleviating the nephrotoxicity of imipenem by cilastatin were investigated in vitro and in rabbits. Our results indicated that imipenem and cilastatin were substrates of hOAT1 and hOAT3. Cilastatin inhibited hOAT1/3-mediated transport of imipenem with IC₅₀ values comparable to the clinical concentration, suggesting the potential to cause a clinical drug–drug interaction (DDI). Moreover, imipenem exhibited hOAT1/3-dependent cytotoxicity, which was alleviated by cilastatin and probenecid. Furthermore, cilastatin and probenecid ameliorated imipenem-induced rabbit acute kidney injury, and reduced the renal secretion of imipenem. Cilastatin and probenecid inhibited intracellular accumulation of imipenem and sequentially decreased the nephrocyte toxicity in rabbit primary proximal tubule cells. Renal OATs, besides DHP-I, was also the target of interaction between imipenem and cilastatin, and contributed to the nephrotoxicity of imipenem. This therefore gives in part the explanation about the mechanism by which cilastatin protected against imipenem-induced nephrotoxicity. Thus, OATs can potentially be used as a therapeutic target to avoid the renal adverse reaction of imipenem in clinic.     

Cancer-specific calcium nanoregulator suppressing the generation and circulation of circulating tumor cell clusters for enhanced anti-metastasis combinational chemotherapy

Tumor metastasis is responsible for chemotherapeutic failure and cancer-related death. Moreover, circulating tumor cell (CTC) clusters play a pivotal role in tumor metastasis. Herein, we develop cancer-specific calcium nanoregulators to suppress the generation and circulation of CTC clusters by cancer membrane-coated digoxin (DIG) and doxorubicin (DOX) co-encapsulated PLGA nanoparticles (CPDDs). CPDDs could precisely target the homologous primary tumor cells and CTC clusters in blood and lymphatic circulation. Intriguingly, CPDDs induce the accumulation of intracellular Ca²⁺ by inhibiting Na⁺/K⁺-ATPase, which help restrain cell–cell junctions to disaggregate CTC clusters. Meanwhile, CPDDs suppress the epithelial–mesenchymal transition (EMT) process, resulting in inhibiting tumor cells escape from the primary site. Moreover, the combination of DOX and DIG at a mass ratio of 5:1 synergistically induces the apoptosis of tumor cells. In vitro and in vivo results demonstrate that CPDDs not only effectively inhibit the generation and circulation of CTC clusters, but also precisely target and eliminate primary tumors. Our findings present a novel approach for anti-metastasis combinational chemotherapy.     

6-Hydroxyindole is an endogenous long-lasting OATP1B1 inhibitor elevated in renal failure patients

The hepatic uptake transporter organic anion transporting polypeptide (OATP) 1B1 is inhibited by some uremic toxins; however, direct inhibition can only partially explain the delayed systemic elimination of substrate drugs in renal failure patients. This study aimed to examine the long-lasting inhibition of OATP1B1 by uremic toxins and their metabolites. Preincubation of HEK293/OATP1B1 cells with 21 uremic toxins resulted in almost no change in the uptake of a typical substrate [³H]estrone-3-sulfate (E₁S), although some directly inhibited [³H]E₁S uptake. In contrast, preincubation with an indole metabolite, 6-hydroxyindole, reduced [³H]E₁S uptake, even after the inhibitor was washed out before [³H]E₁S incubation. Such long-lasting inhibition by 6-hydroxyindole was time-dependent and recovered after a 3-h incubation without 6-hydroxyindole. Preincubation with 6-hydroxyindole increased the Km for [³H]E₁S uptake with minimal change in Vmax. This was compatible with no change in the cell-surface expression of OATP1B1, as assessed by a biotinylation assay. Preincubation with 6-hydroxyindole reduced [³H]E₁S uptake in human hepatocytes without changes in OATP1B1 mRNA. Plasma concentration of 6-hydroxyindole in renal failure patients increased as renal function decreased, but might be insufficient to exhibit potent OATP1B1 inhibition. In conclusion, 6-hydroxyindole is an endogenous long-lasting OATP1B1 inhibitor with elevated plasma concentrations in renal failure patients.     

Novel radioligands for imaging sigma-1 receptor in brain using positron emission tomography (PET)

The sigma-1 receptor (σ₁R) is a unique intracellular protein. σ₁R plays a major role in various pathological conditions in the central nervous system (CNS), implicated in several neuropsychiatric disorders. Imaging of σ₁R in the brain using positron emission tomography (PET) could serve as a noninvasively tool for enhancing the understanding of the disease's pathophysiology. Moreover, σ₁R PET tracers can be used for target validation and quantification in diagnosis. Herein, we describe the radiosynthesis, in vivo PET/CT imaging of novel σ₁R ¹¹C-labeled radioligands based on 6-hydroxypyridazinone, [¹¹C]HCC0923 and [¹¹C]HCC0929. Two radioligands have high affinities to σ₁R, with good selectivity. In mice PET/CT imaging, both radioligands showed appropriate kinetics and distributions. Additionally, the specific interactions of two radioligands were reduced by compounds 13 and 15 (self-blocking). Of the two, [¹¹C]HCC0929 was further investigated in positive ligands blocking studies, using classic σ₁R agonist SA 4503 and σ₁R antagonist PD 144418. Both σ₁R ligands could extensively decreased the uptake of [¹¹C]HCC0929 in mice brain. Besides, the biodistribution of major brain regions and organs of mice were determined in vivo. These studies demonstrated that two radioligands, especially [¹¹C]HCC0929, possessed ideal imaging properties and might be valuable tools for non-invasive quantification of σ₁R in brain.     

2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione,isolated from the root of Averrhoa carambola L., protects against diabetic kidney disease by inhibiting TLR4/TGFβ signaling pathway

ObjectiveDiabetic kidney disease (DKD) is the leading cause of death and disability of diabetes mellitus. However, there is still a lack of specific drugs for the treatment of DKD. The chief aim of this research is to investigate the role and mechanism of 2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD) for DKD.MethodsWild type and TLR4 knockout mice were induced to diabetes. After 4-week treatment with DMDD, blood sugar, renal function, blood lipid and pathological changes were assessed. Real-time PCR, western blotting, and immunohistochemistry were employed to detect the expressions of TLR4, TGFβ1 and Smad2/3 in the renal tissue.ResultsDMDD improved the serum lipid and decreased fasting blood glucose levels in diabetic mice. CysC and urinary albumin levels increased markedly in the diabetic group, and they were obviously decreased after 4 weeks of DMDD treatment. Compared with the WT diabetic mice, the urinary albumin and CysC in the TLR4^-/- mice were expressed at lower levels. HE and Masson’s staining revealed that DMDD clearly ameliorated pathological changes and renal fibrosis. When TLR4 gene was knock out, the pathological was improved. Mechanistically, TLR4, TGF-β1 and Smad2/3 were obvious up-regulation in the renal tissues of diabetic mice. The expressions of these proteins were significantly down-regulated after DMDD treatment (p < 0.05). In the TLR4^-/- mice, mRNA and protein levels of TGF-β1 and Smad2/3 were obviously lower than those in the WT mice. In addition, IHC revealed that a strong in situ expressions of TLR4, TGF-β1 and Smad2/3 were seen in the kidney tissues of diabetic mice, which were distinctly weakened in the DMDD-treated mice. In the TLR4^-/- mice, however, expressions of TGF-β1 and Smad2/3 were not remarkable increase in the diabetic mice compared with normal mice.ConclusionsThese results strongly indicate that TLR4 is essential for DMDD protection against renal dysfunction in diabetic mice. Its hypoglycemic and anti-fibrosis effects were likely mediated by the TLR4/TGFβ signaling pathway.     

Evaluation of 124I-JS001 for hPD1 immuno-PET imaging using sarcoma cell homografts in humanized mice

JS001 (toripalimab) is a humanized IgG monoclonal antibody which strongly inhibits programmed cell death protein 1 (PD1). In this study, we used a different iodine isotype (^nat/124/125I) to label JS001 probes to target the human PD1 (hPD1) antigen. In vitro, the half maximal effective concentration (EC₅₀) value of ^natI-JS001 did not significantly differ from that of JS001. The uptake of ¹²⁵I-JS001 by activated T cells was 5.63 times higher than that by nonactivated T cells after 2 h of incubation. The binding affinity of ¹²⁵I-JS001 to T cells of different lineages after phytohemagglutinin (PHA) stimulation reached 4.26 nmol/L. Humanized PD1 C57BL/6 mice bearing mouse sarcoma S180 cell tumors were validated for immuno-positron emission tomography (immuno-PET) imaging. Pathological staining was used to assess the expression of PD1 in tumor tissues. The homologous ¹²⁴I-human IgG (¹²⁴I-hIgG) group or blocking group was used as a control group. Immuno-PET imaging showed that the uptake in the tumor area of the ¹²⁴I-JS001 group at different time points was significantly higher than that of the blocking group or the ¹²⁴I-hIgG group in the humanized PD1 mouse model. Taken together, these results suggest that this radiotracer has potential for noninvasive monitoring and directing tumor-specific personalized immunotherapy in PD1-positive tumors.     

In situ metabolomics in nephrotoxicity of aristolochic acids based on air flow-assisted desorption electrospray ionization mass spectrometry imaging

Understanding of the nephrotoxicity induced by drug candidates is vital to drug discovery and development. Herein, an in situ metabolomics method based on air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) was established for direct analysis of metabolites in renal tissue sections. This method was subsequently applied to investigate spatially resolved metabolic profile changes in rat kidney after the administration of aristolochic acid I, a known nephrotoxic drug, aimed to discover metabolites associated with nephrotoxicity. As a result, 38 metabolites related to the arginine–creatinine metabolic pathway, the urea cycle, the serine synthesis pathway, metabolism of lipids, choline, histamine, lysine, and adenosine triphosphate were significantly changed in the group treated with aristolochic acid I. These metabolites exhibited a unique distribution in rat kidney and a good spatial match with histopathological renal lesions. This study provides new insights into the mechanisms underlying aristolochic acids nephrotoxicity and demonstrates that AFADESI-MSI-based in situ metabolomics is a promising technique for investigation of the molecular mechanism of drug toxicity.     

Evodiamine-inspired dual inhibitors of histone deacetylase 1 (HDAC1) and topoisomerase 2 (TOP2) with potent antitumor activity

A great challenge in multi-targeting drug discovery is to identify drug-like lead compounds with therapeutic advantages over single target inhibitors and drug combinations. Inspired by our previous efforts in designing antitumor evodiamine derivatives, herein selective histone deacetylase 1 (HDAC1) and topoisomerase 2 (TOP2) dual inhibitors were successfully identified, which showed potent in vitro and in vivo antitumor potency. Particularly, compound 30a was orally active and possessed excellent in vivo antitumor activity in the HCT116 xenograft model (TGI = 75.2%, 150 mg/kg, p.o.) without significant toxicity, which was more potent than HDAC inhibitor vorinostat, TOP inhibitor evodiamine and their combination. Taken together, this study highlights the therapeutic advantages of evodiamine-based HDAC1/TOP2 dual inhibitors and provides valuable leads for the development of novel multi-targeting antitumor agents.     

In vitro inhibitory effects of components from Salvia miltiorrhiza on catalytic activity of three human Arachidonic acid ω-hydroxylases

CYP4 enzymes are involved in the metabolism of xenobiotics and endogenous molecules. 20-Hydroxyeicosatetraenoic acid (20-HETE), the arachidonic acid (AA) ω-hydroxylation metabolite catalyzed by CYP4A/4F enzymes, is implicated in various biological functions. The goal of this investigation is to examine the inhibitory effects of components from Salvia miltiorrhiza(Danshen) on AA ω-hydroxylation using recombinant CYP4A11, CYP4F2, CYP4F3B, and microsomal systems. Tanshinone IIA had noncompetitive inhibition on CYP4F3B (K_i = 4.98 μM). Cryptotanshinone (K_i = 6.87 μM) and tanshinone I (K_i = 0.42 μM) had mixed-type inhibition on CYP4A11. Dihydrotanshinone I had mixed-type inhibition on CYP4A11 (K_i = 0.09 μM), and noncompetitive inhibition on CYP4F2 (K_i = 4.25 μM) and CYP4F3B (K_i = 3.08 μM). Salvianolic acid A had competitive inhibition on CYP4A11 (K_i = 19.37 μM), and noncompetitive inhibition on CYP4F2 (K_i = 15.28 μM) and CYP4F3B (K_i = 6.45 μM). Salvianolic acid C had noncompetitive inhibition on CYP4F2 (K_i = 5.70 μM) and CYP4F3B (K_i = 18.64 μM). In human kidney, human liver or rat heart microsomes, 20-HETE formation was significantly inhibited (P < 0.05) by dihydrotanshinone I (5 and 20 μM) and salvianolic acid A (20 and 50 μM). Given that low plasma concentrations of Danshen components after oral administration, Danshen preparations may not play pharmacological roles by inhibiting AA ω-hydroxylases; however, as Danshen components may reach high concentration in human intestine, drugs that have an important pre-systemic metabolism by these CYP4A/4F enzymes should avoid being co-administered with Danshen preparations.     

The protective effects of silymarin nanoemulsion on 5-fluorouracil-induced gastrointestinal toxicity in rats

5-Fluorouracil (5FUra) is the third most popular chemotherapeutic component employed to treat solid tumors. In the present study, we aimed to appraise the silymarin (SM) and silymarin nanoemulsion (SMN) effect on 5FUra-induced gastrointestinal toxicity in adult male rats. A total of 30 male Wistar rats were divided into 6 groups including the control (Crl) group, and groups treated with SMN (5 mg.kg⁻¹), SM (5 mg.kg⁻¹), 5FUra + SMN (5 mg.kg⁻¹), and 5FUra + SM (5 mg.kg⁻¹) by IP injection for 14 days. And gastrointestinal toxicity was induced by a single intraperitoneal (IP) injection of 5FUra (100 mg.kg⁻¹) for the last group in the study. Treating rats with SM and SMN diminished elevating malondialdehyde (MDA) levels, and improved total antioxidant capacity (TAC) levels. Also, the intensity of mRNA expression of interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) caused by 5FUra in the gastrointestinal tissue tract, and macroscopic oral ulcerations decreased, ass well as weight loss was prevented, particularly in the SMN group. Moreover, in the microscopic scope, there were significant improvements in the levels of hyperemia, hyaline, and inflammatory cell infiltration in the tongue, esophagus, and intestinal tissues in the FUra + SMN and FUra + SM groups compared to 5FUra. Hence, treatment with SM and SMN reduced oxidative stress, histopathological degeneration, and gene expression of inflammatory markers in the gastrointestinal tract. According to the results, treatment with SM and SMN markedly decreases the gastrointestinal toxicity caused by 5FUra.