Carvedilol alleviates the detrimental effects of azathioprine on hepatic tissues in experimental rats: Focusing on redox system, inflammatory and apoptosis pathways.

PURPOSE: Drug-induced liver injury is becoming an increasingly important topic in drug research and clinical practice. Due to a lack of experimental animal models, predicting drug-induced liver injury in humans is challenging. Azathioprine (AZA) is a classical immunosuppressant with hepatotoxic adverse effects. The present study aimed to address the hepatoprotective effect of carvedilol (CAR) against AZA-induced hepatocellular injury via assessing redox-sensitive signals. METHOD: To achieve this purpose, rats were allocated into four groups: control, CAR only, AZA only, and CAR plus AZA groups. The induction of hepatic injury was induced by a single intraperitoneal injection of AZA at a dose of 50 mg/kg on the 6th day of the experiment. Each experimental protocol was approved and supervised by the Ethics Committee for Animal Experiments. RESULTS: The results of the present study revealed that CAR administration significantly diminished AZA-induced hepatic dysfunction, as evidenced by relief of hepatic function biomarkers and histopathological aberration induced by AZA injection. Besides, CAR restored oxidant/antioxidant balance as well as NRF2 expression. In addition, CAR suppressed inflammatory response induced by AZA challenge as evidenced by downregulation of TLR4, TNF-α, MPO, and eNOS/iNOS levels in hepatic tissue. Moreover, CAR recovered apoptotic/anti-apoptotic status by modulation of caspase-3/Bcl2 expression. CONCLUSION: Taken together, CAR protects against AZA-induced hepatic injury via antioxidant, anti-inflammatory, and anti-apoptotic activities. These findings revealed that CAR could be a good candidate for hepatic injury protection and can be added to AZA therapeutic regimen to reduce their adverse effect.

Gut microbiota biotransformation of drug glucuronides leading to gastrointestinal toxicity: Therapeutic potential of bacterial ß-glucuronidase inhibition in mycophenolate-induced enteropathy.

AIMS: Drug-induced enteropathy is often associated with the therapeutic use of certain glucuronidated drugs. One such drug is mycophenolic acid (MPA), a well-established immunosuppressant of which gastrointestinal adverse effects are a major concern. The role of bacterial ß-glucuronidase (ß-G) from the gut microbiota in MPA-induced enteropathy has recently been discovered. Bacterial ß-G hydrolyzes MPAG, the glucuronide metabolite of MPA excreted in the bile, leading to the digestive accumulation of MPA that would favor in turn these adverse events. We therefore hypothesized that taming bacterial ß-G activity might reduce MPA digestive exposure and prevent its toxicity. MAIN METHODS: By using a multiscale approach, we evaluated the effect of increasing concentrations of MPA on intestinal epithelial cells (Caco-2 cell line) viability, proliferation, and migration. Then, we investigated the inhibitory properties of amoxapine, a previously described bacterial ß-G inhibitor, by using molecular dynamics simulations, and evaluated its efficiency in blocking MPAG hydrolysis in an Escherichia coli-based ß-G activity assay. The pharmacological effect of amoxapine was evaluated in a mouse model. KEY FINDINGS: We observed that MPA impairs intestinal epithelial cell homeostasis. Amoxapine efficiently blocks the hydrolysis of MPAG to MPA and significantly reduces digestive exposure to MPA in mice. As a result, administration of amoxapine in MPA-treated mice significantly attenuated gastrointestinal lesions. SIGNIFICANCE: Collectively, these results suggest that the digestive accumulation of MPA is involved in the pathophysiology of MPA-gastrointestinal adverse effects. This study provides a proof-of-concept of the therapeutic potential of bacterial ß-G inhibitors in glucuronidated drug-induced enteropathy.

Synergistic Renoprotective Effects of Green Tea Extract and Gemigliptin against Tacrolimus-Induced Nephrotoxicity in Mice / Efectos Renoprotectores Sinérgicos del Extracto de Té Verde y Gemigliptina contra la Nefrotoxicidad Inducida por Tacrolimus en Ratones

SUMMARY: Although tacrolimus (TAC) significantly reduces allograft rejection incidence in solid-organ transplantation, its long-term use is associated with an increased risk of TAC-induced nephrotoxicity. In this study, we investigated the renoprotective effects of green tea extract (GTE) with or without the dipeptidyl peptidase 4 inhibitor, gemigliptin, by assessing serum creatinine levels, the amount of proteinuria, and histopathology in TAC-induced nephrotoxicity. TAC-induced nephrotoxicity was induced by intraperitoneal TAC injection, GTE was administered via subcutaneous injection, and gemigliptin was administered orally. Mice with TAC-induced nephrotoxicity exhibited a significant increase in both serum creatinine levels and 24-hour urine protein. However, when treated with GTE via subcutaneous injection, mice showed a decrease in serum creatinine levels and the amount of proteinuria. When GTE was combined with gemigliptin, further renoprotective effects were observed in biochemical assessments, consistent with the attenuation of TAC-induced nephrotoxicity in histopathology. The expression of p53 protein was lower in the mice treated with the combination of GTE and gemigliptin compared to mice with TAC-induced nephrotoxicity. Our results demonstrate that the combination of GTE and gemigliptin treatment reveals synergistic renoprotective effects by decreasing the expression of p53 protein. These findings suggest that the combination of GTE and gemigliptin could potentially be used as a prophylactic or therapeutic strategy for TAC-induced nephrotoxicity.

Aunque tacrolimus (TAC) reduce significativamente la incidencia de rechazo de aloinjertos en trasplantes de órganos sólidos, su uso a largo plazo se asocia con un mayor riesgo de nefrotoxicidad inducida por TAC. En este estudio, investigamos los efectos renoprotectores del extracto de té verde (GTE) con o sin el inhibidor de la dipeptidil peptidasa 4, gemigliptina, mediante la evaluación de los niveles de creatinina sérica, la cantidad de proteinuria y la histopatología en la nefrotoxicidad inducida por TAC. La nefrotoxicidad inducida por TAC se indujo mediante inyección intraperitoneal de TAC, el GTE se administró mediante inyección subcutánea y la gemigliptina se administró por vía oral. Los ratones con nefrotoxicidad inducida por TAC mostraron un aumento significativo tanto en los niveles de creatinina sérica como en la proteína en orina de 24 horas. Sin embargo, cuando se trataron con GTE mediante inyección subcutánea, los ratones mostraron una disminución en los niveles de creatinina sérica y en la cantidad de proteinuria. Cuando se combinó GTE con gemigliptina, se observaron efectos renoprotectores adicionales en las evaluaciones bioquímicas, lo que concuerda con la atenuación de la nefrotoxicidad inducida por TAC en histopatología. La expresión de la proteína p53 fue menor en los ratones tratados con la combinación de GTE y gemigliptina en comparación con los ratones con nefrotoxicidad inducida por TAC. Nuestros resultados demuestran que la combinación de tratamiento con GTE y gemigliptina revela efectos renoprotectores sinérgicos al disminuir la expresión de la proteína p53. Estos hallazgos sugieren que la combinación de GTE y gemigliptina podría usarse potencialmente como estrategia profiláctica o terapéutica para la nefrotoxicidad inducida por TAC.

Cyclosporine-A induced cytotoxicity within HepG2 cells by inhibiting PXR mediated CYP3A4/CYP3A5/MRP2 pathway.

Cyclosporine-A (CsA) is currently used to treat immune rejection after organ transplantation as a commonly used immunosuppressant. Liver injury is one of the most common adverse effects of CsA, whose precise mechanism has not been fully elucidated. Pregnane X receptor (PXR) plays a critical role in mediating drug-induced liver injury as a key regulator of drug and xenobiotic clearance. As a nuclear receptor, PXR transcriptionally upregulates the expression of drug-metabolizing enzymes and drug transporters, including cytochrome P4503A (CPY3A) and multidrug resistance-associated protein 2 (MRP2). Our study established CsA-induced cytotoxic hepatocytes in an in vitro model, demonstrating that CsA dose-dependently increased the aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) level secreted in the HepG2 cell supernatant, as well as viability and oxidative stress of HepG2 cells. CsA also dose-dependently decreased the PXR, CYP3A4, CPY3A5, and MRP2 levels of HepG2 cells. Mechanistically, altering the expression of PXR, CYP3A4, CYP3A5, and MRP2 affected the impact of CsA on AST and LDH levels. Moreover, altering the expression of PXR also changed the level of CYP3A4, CPY3A5, and MRP2 of HepG2 cells treated by CsA. Our presented findings provide experimental evidence that CsA-induced liver injury is PXR tightly related. We suggest that PXR represents an attractive target for therapy of liver injury due to its central role in the regulation of the metabolizing enzymes CYP3A and MRP2-mediated bile acid transport and detoxification.

Caragana sinica (Buc'hoz) Rehd. (jin ji er) polysaccharide regulates the immune function and intestinal microbiota of cyclophosphamide (CTX) induced immunosuppressed mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Caragana sinica (Buc'hoz) Rehd. is a plant widely grown in Yunnan, China, for both medicinal and edible purposes. The "National Compilation of Chinese Herbal Medicine" describes its nature as "slightly temperate and sweet". Caragana sinica is usually medicated with whole herbs, the main function is to replenish the kidneys and stop bleeding. Caragana sinica was used in folk medicine in Chuxiong, Yunnan, to treat deficiency colds, fatigue, fever, cough, hypertension, and other diseases. AIM OF THE STUDY: This article investigates the structural characteristics of Caragana sinica polysaccharide (CSP) and explores its immune-regulatory activity and molecular biological mechanisms in cyclophosphamide-induced immunosuppressed mice, as well as its effects on intestinal bacteria. METHODS: With the water-extraction and alcohol-precipitation method, Caragana sinica polysaccharide were extracted, obtaining CSP by purification. A variety of methods and techniques have been used to analyze the chemical properties and structural characteristics of CSP. Immunosuppressive mice model was established through intraperitoneal injection of cyclophosphamide (CTX) to study the immune-regulatory effects and mechanisms of CSP. RESULTS: The data indicated that CSP is a neutral heteropolysaccharide mainly composed of arabinose and galactose. This article uses immunosuppressive mice induced by cyclophosphamide (CTX) as the model. The results showed that CSP can promote the immune function of CTX treated immunosuppressed mice and regulate the diversity and composition of intestinal microbiota. CSP can increase macrophage phagocytosis, NK cell killing activity, and lymphocyte proliferation activity. It can also repair the index and morphological damage of the thymus and spleen. And by binding to the TLR4 receptor, MyD88 was activated and interacted with TRAF6 to promote the transfer of NF-κB into the nucleus. Thereby promoting cytokine release and increasing the production of IL-1ß, IL-6, IL-10, TNF-α, IgA, and IgG in the serum. CSP also effectively alleviated the liver damage caused by CTX through antioxidant activity. Furthermore, CSP can dramatically affect the intestinal microbiota and the body's immunity by boosting the relative presence of Bacteroides and Verrucamicrobiota. CONCLUSIONS: Research results indicated that CSP can regulate the immune function of mice, providing a basis for developing CSP as a potential immune modulator and functional food.

Metabolic network and proteomic expression perturbed by cyclosporine A to model microbe Escherichia coli.

Cyclosporine A (CsA) is a model drug that has caused great concern due to its widespread use and abuse in the environment. However, the potential harm of CsA to organisms also remains largely unknown, and this issue is exceptionally important for the health risk assessment of antibiotics. To address this concern, the crosstalk between CsA stress and cellular metabolism at the proteomic level in Escherichia coli was investigated and dissected in this study. The results showed that CsA inhibited E. coli growth in a time-dependent manner. CsA induced reactive oxygen species (ROS) overproduction in a dose- and time-dependent manner, leading to membrane depolarization followed by cell apoptosis. In addition, translation, the citric acid cycle, amino acid biosynthesis, glycolysis and responses to oxidative stress and heat were the central metabolic pathways induced by CsA stress. The upregulated proteins, including PotD, PotF and PotG, controlled cell growth. The downregulated proteins, including SspA, SspB, CstA and DpS, were regulators of self-feedback during the starvation process. And the up- and downregulated proteins, including AtpD, Adk, GroS, GroL and DnaK, controlled energy production. These results provide an important reference for the environmental health risk assessment of CsA.

Toxic effects of sirolimus and everolimus on the development and behavior of zebrafish embryos.

Sirolimus and everolimus have been widely used in children. These mammalian target of rapamycin (mTOR) inhibitors have shown excellent efficacy not only in organ transplant patients as immunosuppressive agents but also in patients with some other diseases. However, whether mTOR inhibitors can affect the growth and development of children is of great concern. In this study, using zebrafish models, we discovered that sirolimus and everolimus could slow the development of zebrafish, affecting indicators such as survival, hatching, deformities, body length, and movement. In addition to these basic indicators, sirolimus and everolimus had certain slowing effects on the growth and development of the nervous system, blood vessels, and the immune system. These effects were dose dependent. When the drug concentration reached or exceeded 0.5 µM, the impacts of sirolimus and everolimus were very significant. More interestingly, the impact was transient. Over time, the various manifestations of experimental embryos gradually approached those of control embryos. We also compared the effects of sirolimus and everolimus on zebrafish, and we revealed that there was no significant difference between these drugs in terms of their effects. In summary, the dose of sirolimus and everolimus in children should be strictly controlled, and the drug concentration should be monitored over time. Otherwise, drug overdosing may have a certain impact on the growth and development of children.

Physiologically based pharmacokinetic model combined with reverse dose method to study the nephrotoxic tolerance dose of tacrolimus.

Nephrotoxicity is the most common side effect that severely limits the clinical application of tacrolimus (TAC), an immunosuppressive agent used in kidney transplant patients. This study aimed to explore the tolerated dose of nephrotoxicity of TAC in individuals with different CYP3A5 genotypes and liver conditions. We established a human whole-body physiological pharmacokinetic (WB-PBPK) model and validated it using data from previous clinical studies. Following the injection of 1 mg/kg TAC into the tail veins of male rats, we developed a rat PBPK model utilizing the drug concentration-time curve obtained by LC-MS/MS. Next, we converted the established rat PBPK model into the human kidney PBPK model. To establish renal concentrations, the BMCL5 of the in vitro CCK-8 toxicity response curve (drug concentration range: 2-80 mol/L) was extrapolated. To further investigate the acceptable levels of nephrotoxicity for several distinct CYP3A5 genotypes and varied hepatic function populations, oral dosing regimens were extrapolated utilizing in vitro-in vivo extrapolation (IVIVE). The PBPK model indicated the tolerated doses of nephrotoxicity were 0.14-0.185 mg/kg (CYP3A5 expressors) and 0.13-0.155 mg/kg (CYP3A5 non-expressors) in normal healthy subjects and 0.07-0.09 mg/kg (CYP3A5 expressors) and 0.06-0.08 mg/kg (CYP3A5 non-expressors) in patients with mild hepatic insufficiency. Further, patients with moderate hepatic insufficiency tolerated doses of 0.045-0.06 mg/kg (CYP3A5 expressors) and 0.04-0.05 mg/kg (CYP3A5 non-expressors), while in patients with moderate hepatic insufficiency, doses of 0.028-0.04 mg/kg (CYP3A5 expressors) and 0.022-0.03 mg/kg (CYP3A5 non-expressors) were tolerated. Overall, our study highlights the combined usage of the PBPK model and the IVIVE approach as a valuable tool for predicting toxicity tolerated doses of a drug in a specific group.

A multi-omics investigation of tacrolimus off-target effects on a proximal tubule cell-line.

INTRODUCTION: Tacrolimus, an immunosuppressive drug prescribed to a majority of organ transplant recipients is nephrotoxic, through still unclear mechanisms. This study on a lineage of proximal tubular cells using a multi-omics approach aims to detect off-target pathways modulated by tacrolimus that can explain its nephrotoxicity. METHODS: LLC-PK1 cells were exposed to 5 µM of tacrolimus for 24 h in order to saturate its therapeutic target FKBP12 and other high-affine FKBPs and favour its binding to less affine targets. Intracellular proteins and metabolites, and extracellular metabolites were extracted and analysed by LC-MS/MS. The transcriptional expression of the dysregulated proteins PCK-1, as well as of the other gluconeogenesis-limiting enzymes FBP1 and FBP2, was measured using RT-qPCR. Cell viability with this concentration of tacrolimus was further checked until 72 h. RESULTS: In our cell model of acute exposure to a high concentration of tacrolimus, different metabolic pathways were impacted including those of arginine (e.g., citrulline, ornithine) (p < 0.0001), amino acids (e.g., valine, isoleucine, aspartic acid) (p < 0.0001) and pyrimidine (p < 0.01). In addition, it induced oxidative stress (p < 0.01) as shown by a decrease in total cell glutathione quantity. It impacted cell energy through an increase in Krebs cycle intermediates (e.g., citrate, aconitate, fumarate) (p < 0.01) and down-regulation of PCK-1 (p < 0.05) and FPB1 (p < 0.01), which are key enzymes in gluconeogenesis and acid-base balance control. DISCUSSION: The variations found using a multi-omics pharmacological approach clearly point towards a dysregulation of energy production and decreased gluconeogenesis, a hallmark of chronic kidney disease which may also be an important toxicity pathway of tacrolimus.

Morphology of the male rat gonad after in Utero exposure to immunosuppressants.

The protocol for immunosuppression of pregnant women is based on immunosuppressant panels. The aim of the study was to determine the influence of commonly applied combinations of immunosuppressants to pregnant rats on the morphology of the offspring' testes. Pregnant rats were treated with cyclosporin A (CsA), mycophenolate mofetil (MMF) and prednisone (Pred) (CMG); tacrolimus (Tc), MMF and Pred (TMG); CsA, everolimus (Ev) and Pred (CEG). Testes of mature offspring underwent morphological analysis. Mainly in the testes of CMG and TMG rats the morphological and functional changes were observed: immature germ cells (GCs) in the seminiferous tubule (ST) lumen, invaginations of the basement membrane, infolding to the seminiferous epithelium (SE), the ST wall thickening, increased acidophilia of Sertoli cells' (SCs) cytoplasm, large residual bodies near the lumen, dystrophic ST and tubules look like the Sertoli cell-only syndrome, Leydig cells with abnormal cell nucleus, hypertrophy of the interstitium, blurring of the boundary between ST wall and interstitium, a reduced number of GCs in the SE, vacuolation of the SE. In the CEG there were only a reduced number of GCs in some tubules and vacuolization of SCs. The safest combination of drugs was CEG, while the TMG and CMG were gonadotoxic.