Protective effect of SRT1720 on acute liver injury induced by acetaminophen in mice and its mechanism / 中国生物制品学杂志

@#Objective To evaluate the protective effect of the activator of silent information regulator 2-related enzymes 1(SIRT1),SRT1720,on liver injury induced by acetaminophen(APAP)in mice and explore its mechanism. Methods Forty male C57BL/6J mice were randomly divided into normal control group,SRT1720 treatment group,APAP treatment group,and APAP + SRT1720 treatment group,with 10 mice in each group. Mice in SRT1720 and APAP + SRT1720 groups were given SRT1720(30 mg/kg body mass)by intragastric administration,while normal saline of equal volume was given by intragastric administration in control and APAP groups,once a day for 5 days;On the 6th day,mice in APAP and APAP + SRT1720 groups were injected i. p. with APAP(325 mg/kg body mass),while those in control and SRT1720 groups with equal volume of normal saline. After 24 hours,the peripheral blood was taken and the serum was separated,which were detected for the levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)by the corresponding kits;The liver tissue of mice was taken aseptically,observed for the pathological changes by HE staining,detected for the mRNA transcription levels of GRP78,PERK,eIF2 α,ATF4 and CHOP genes related to PERK-eIF2α-CHOP signaling pathway by RT-qPCR and detected for the relative expression levels of these corresponding proteins and Caspase12 protein by Western blot. Results Compared with normal control group,the serum ALT and AST levels of mice in APAP group significantly increased(t = 55. 21 and34. 29 respectively,each P < 0. 01);significant necrosis of hepatocytes was observed in liver tissue,the structure of hepatic lobules changed significantly,and the swelling and deformation of hepatocytes in some areas were serious;the mRNA transcription and relative protein expression levels of GRP78,PERK,eIFα,ATF4 and CHOP genes increased significantly(t = 9. 85～33. 89,each P < 0. 05)and the relative expression level of Caspase12 protein increased significantly(t = 11. 78,P < 0. 01). Compared with APAP group,the serum ALT and AST levels of mice in APAP + SRT1720 group decreased significantly(t = 42. 92 and 18. 02 respectively,each P < 0. 01);the degree of hepatocyte injury was obviously reduced and the number of swollen and deformed cells also significantly decreased;the mRNA transcription and relative protein expression levels of GRP78,PERK,eIF2α,ATF4 and CHOP decreased significantly(t = 6. 19～22. 43,each P < 0. 05)and the expression level of Caspase12 protein showed no significant decrease(t = 0. 34,P > 0. 05). Conclusion SRT1720improved APAP-induced liver injury in mice,possibly by inhibiting PERK-eIF2α-CHOP signaling pathway in endoplasmic reticulum stress(ERS).

Antioxidant mediated defensive potency of Caesalpinia bonducella nut on Acetaminophen-inebriated spleen and cardiotoxicity: Implications on oxidative stress and tissue morphology in an In vivo model

Overdosing on medications can be unintentional or deliberate. Acetaminophen (APAP) is a widely used over-the-counter analgesic and antipyretic drug. APAP overdose can induce spleen and cardiotoxicity apart from hepatotoxicity. Bonduc nut is well-known for its medicinal and therapeutic properties. More scientific data is necessary to be therapeutically relevant. This study examined the effects of Bonduc nut extract (BNE) on APAP-induced spleen and cardiotoxicity in Wistar albino rats. The rats were divided into five groups of six rats each. In vitro assays were carried out to analyze antioxidant activity and free radical scavenging activity in aqueous, ethanol, and methanol solvents in Bonduc nut powder. Total phenolic content, DPPH, catalase, and peroxidase activity were used to test antioxidant activity. The rats were euthanized after the study period to examine antioxidant parameters such as superoxide dismutase, catalase, reduced glutathione, and glutathione peroxidase, as well as lipid peroxidation and histopathology of the spleen and heart tissues. Results suggest that compared to other solvents aqueous has better Invitro antioxidant ability and the same extract significantly increased the antioxidant and reduced lipid peroxidation followed by restoring the tissue morphology in APAP-induced spleen and cardiotoxicity. The outcome of the study revealed that aqueous BNE has a significant protective efficacy against APAP-induced spleen and cardiotoxicity in Wistar albino rats.

Fluorine-thiol displacement probes for acetaminophen's hepatotoxicity

Chemicals possessing reactive electrophiles can denature innate proteins leading to undesired toxicity, and the overdose-induced liver injury by drugs containing electrophiles has been one of the major causes of non-approval and withdraw by the US Food and Drug Administration (FDA). Elucidating the associated proteins could guide the future development of therapeutics to circumvent these drugs' toxicities, but was largely limited by the current probing tools due to the steric hindrance of chemical tags including the common "click chemistry" labels. Taking the widely used non-steroidal anti-inflammatory drug acetaminophen (APAP) as an example, we hereby designed and synthesized an APAP analogue using fluorine as a steric-free label. Cell toxicity studies indicated our analogue has similar activity to the parent drug. This analogue was applied to the mouse hepatocellular proteome together with the corresponding desthiobiotin-SH probe for subsequent fluorine-thiol displacement reactions (FTDRs). This set of probes has enabled the labeling and pull-down of hepatocellular target proteins of the APAP metabolite as validated by Western blotting. Our preliminary validation results supported the interaction of APAP with the thioredoxin protein, which is an important redox protein for normal liver function. These results demonstrated that our probes confer minimal steric perturbation and mimic the compounds of interest, allowing for global profiling of interacting proteins. The fluorine-thiol displacement probing system could emerge as a powerful tool to enable the investigation of drug-protein interactions in complex biological environments.

SIRT6 as a key event linking P53 and NRF2 counteracts APAP-induced hepatotoxicity through inhibiting oxidative stress and promoting hepatocyte proliferation

Acetaminophen (APAP) overdose is the leading cause of drug-induced liver injury, and its prognosis depends on the balance between hepatocyte death and regeneration. Sirtuin 6 (SIRT6) has been reported to protect against oxidative stress-associated DNA damage. But whether SIRT6 regulates APAP-induced hepatotoxicity remains unclear. In this study, the protein expression of nuclear and total SIRT6 was up-regulated in mice liver at 6 and 48 h following APAP treatment, respectively.

Dual roles of p62/SQSTM1 in the injury and recovery phases of acetaminophen-induced liver injury in mice

Acetaminophen (APAP) overdose can induce liver injury and is the most frequent cause of acute liver failure in the United States. We investigated the role of p62/SQSTM1 (referred to as p62) in APAP-induced liver injury (AILI) in mice. We found that the hepatic protein levels of p62 dramatically increased at 24 h after APAP treatment, which was inversely correlated with the hepatic levels of APAP-adducts. APAP also activated mTOR at 24 h, which is associated with increased cell proliferation. In contrast, p62 knockout (KO) mice showed increased hepatic levels of APAP-adducts detected by a specific antibody using Western blot analysis but decreased mTOR activation and cell proliferation with aggravated liver injury at 24 h after APAP treatment. Surprisingly, p62 KO mice recovered from AILI whereas the wild-type mice still sustained liver injury at 48 h. We found increased number of infiltrated macrophages in p62 KO mice that were accompanied with decreased hepatic von Willebrand factor (VWF) and platelet aggregation, which are associated with increased cell proliferation and improved liver injury at 48 h after APAP treatment. Our data indicate that p62 inhibits the late injury phase of AILI by increasing autophagic selective removal of APAP-adducts and mitochondria but impairs the recovery phase of AILI likely by enhancing hepatic blood coagulation.

Recommendations for the use of the acetaminophen hepatotoxicity model for mechanistic studies and how to avoid common pitfalls

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, which is safe at therapeutic doses but can cause severe liver injury and even liver failure after overdoses. The mouse model of APAP hepatotoxicity recapitulates closely the human pathophysiology. As a result, this clinically relevant model is frequently used to study mechanisms of drug-induced liver injury and even more so to test potential therapeutic interventions. However, the complexity of the model requires a thorough understanding of the pathophysiology to obtain valid results and mechanistic information that is translatable to the clinic. However, many studies using this model are flawed, which jeopardizes the scientific and clinical relevance. The purpose of this review is to provide a framework of the model where mechanistically sound and clinically relevant data can be obtained. The discussion provides insight into the injury mechanisms and how to study it including the critical roles of drug metabolism, mitochondrial dysfunction, necrotic cell death, autophagy and the sterile inflammatory response. In addition, the most frequently made mistakes when using this model are discussed. Thus, considering these recommendations when studying APAP hepatotoxicity will facilitate the discovery of more clinically relevant interventions.

Protective effects of extracts of Schisandra chinensis stems against acetaminophen-induced hepatotoxicity via regulation of MAPK and caspase-3 signaling pathways / 中国天然药物

The present study was designed to evaluate protective activity of an ethanol extract of the stems of Schisandra chinensis (SCE) and explore its possible molecular mechanisms on acetaminophen (APAP) induced hepatotoxicity in a mouse model. The results of HPLC analysis showed that the main components of SCE included schisandrol A, schisandrol B, deoxyschisandrin, schisandrin B, and schisandrin C and their contents were 5.83, 7.11, 2.13, 4.86, 0.42 mg·g, respectively. SCE extract was given for 7 consecutive days before a single hepatotoxic dose of APAP (250 mg·kg) was injected to mice. Our results showed that SCE pretreatment ameliorated liver dysfunction and oxidative stress, which was evidenced by significant decreases in aspartate transaminase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA) contents and elevations in reduced glutathione (GSH) and superoxide dismutase (SOD) levels. These findings were associated with the result that the SCE pretreatment significantly decreased expression levels of 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT). SCE also significantly decreased the expression levels of Bax, mitogen- activated protein kinase (MAPK), and cleaved caspase-3 by APAP exposure. Furthermore, supplementation with SCE suppressed the expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), suggesting alleviation of inflammatory response. In summary, these findings from the present study clearly demonstrated that SCE exerted significant alleviation in APAP-induced oxidative stress, inflammation and apoptosis mainly via regulating MAPK and caspase-3 signaling pathways.

Protective effect of Fuzheng Yanggan Mixture on drug-induced liver injury / 中国中药杂志

The model of drug-induced liver injury (DILI) induced by acetaminophen (APAP) in mice was established to investigate the anti-oxidation and anti-ferroptosis mechanisms of Fuzheng Yanggan Mixture on DILI. C57BL/6 mice were randomly divided into five groups: control group, model group, positive group, and low and high-dose Fuzheng Yanggan Mixture groups (0.12, 0.24 g·kg⁻¹). Mice were intragastrically administration with Fuzheng Yanggan Mixture (0.12, 0.24 g·kg⁻¹) once per day for 21 consecutive days, and at the same time, mice were weighted every day. The mice were injected intraperitoneally with 600 mg·kg⁻¹ of APAP to establish a mouse model of acute DILI after 16 h from the last administration of Fuzheng Yanggan Mixture. After 6 h from APAP challenge, the experimental animals were weighted and sacrificed to collect blood and liver tissue samples. And then, the effect of Fuzheng Yanggan Mixture on liver weight and the liver weight ratio of mice were examined; the content of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum and the level of malondialdehyde (MDA), glutathione (GSH) and nicotinamide adenine dinucleotide phosphate (NADPH) in the liver tissue were measured. Prostaglandinendoperoxide synthase 2(ptgs2) mRNA level in liver tissues was detected by Q-PCR, and protein expression levels of SLC7A11 and GPX4 in liver tissues were detected by Western blot. Moreover, HE staining, immunohistochemical assay and TUNEL staining were used to observe pathological changes of the liver tissue sections. It is found that Fuzheng Yanggan Mixture could relieve APAP-induced liver enlargement and inhibit hepatic weight ratio increase. Compared with model group, the mice in Fuzheng Yanggan Mixture groups showed decreases in the content of ALT, AST and MDA, and increases in the content of GSH and NADPH. What is more, Fuzheng Yanggan Mixture could down-regulate ptgs2 mRNA level and up-regulate SLC7A11 and GPX4 protein levels. All of the results lead to a conclusion that Fuzheng Yanggan Mixture plays a protective effect on DILI in mice, which may be associated with the inhibition of ferroptosis.

Protective effects of extracts of Schisandra chinensis stems against acetaminophen-induced hepatotoxicity via regulation of MAPK and caspase-3 signaling pathways / 中国天然药物

The present study was designed to evaluate protective activity of an ethanol extract of the stems of Schisandra chinensis (SCE) and explore its possible molecular mechanisms on acetaminophen (APAP) induced hepatotoxicity in a mouse model. The results of HPLC analysis showed that the main components of SCE included schisandrol A, schisandrol B, deoxyschisandrin, schisandrin B, and schisandrin C and their contents were 5.83, 7.11, 2.13, 4.86, 0.42 mg·g, respectively. SCE extract was given for 7 consecutive days before a single hepatotoxic dose of APAP (250 mg·kg) was injected to mice. Our results showed that SCE pretreatment ameliorated liver dysfunction and oxidative stress, which was evidenced by significant decreases in aspartate transaminase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA) contents and elevations in reduced glutathione (GSH) and superoxide dismutase (SOD) levels. These findings were associated with the result that the SCE pretreatment significantly decreased expression levels of 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT). SCE also significantly decreased the expression levels of Bax, mitogen- activated protein kinase (MAPK), and cleaved caspase-3 by APAP exposure. Furthermore, supplementation with SCE suppressed the expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), suggesting alleviation of inflammatory response. In summary, these findings from the present study clearly demonstrated that SCE exerted significant alleviation in APAP-induced oxidative stress, inflammation and apoptosis mainly via regulating MAPK and caspase-3 signaling pathways.

Continuous positive airway pressure therapy: New generations.

Continuous positive airway pressure (CPAP) is the treatment of choice for obstructive sleep apnoea syndrome (OSAS). However, CPAP is not tolerated by all patients with OSAS and alternative modes of pressure delivery have been developed to overcome pressure intolerance, thereby improving patient comfort and adherence. Auto-adjustable positive airway pressure (APAP) devices may be utilised for the long-term management of OSAS and may also assist in the initial diagnosis of OSAS and titration of conventional CPAP therapy. Newer modalities such as C-Flex and A-Flex also show promise as treatment options in the future. However, the evidence supporting the use of these alternative modalities remains scant, in particular with regard to long-term cardiovascular outcomes. In addition, not all APAP devices use the same technological algorithms and data supporting individual APAP devices cannot be extrapolated to support all. Further studies are required to validate the roles of APAP, C-Flex and AFlex. In the interim, standard CPAP therapy should continue as the mainstay of OSAS management.

Titulación domiciliaria de CPAP en el síndrome de apnea del sueño: ¿una, 3 ó 7 noches? / Home auto-titrating positive airway (APAP) for sleep apnea syndrome: one, three or seven nights?

Los equipos automáticos de CPAP (APAP) permiten establecer la presión eficaz de tratamiento en pacientes con apnea del sueño, sin la necesidad de supervisión y con un menor costo que las titulaciones realizadas bajo control polisomnográfico. Entre estos, el Autoset (ResMed) modifica la presión entregada en base a la presencia de apneas y limitación al flujo inspiratorio. Pacientes con síndrome de apnea/hipopnea del sueño (SAHS) fueron estudiados con este equipo durante 1 semana, luego de un período de al menos 60 días de uso de CPAP con una presión arbitraria entre 8 y 9 cmH2O. Comparamos el valor de presión obtenido en 1, 3 y 7 noches de titulación. Se estableció un cumplimiento mínimo aceptable de 6 noches y un nivel de fuga promedio < 0.4 l/seg. Ingresaron al estudio 24 pacientes de los cuales se excluyeron 2 (uno por fuga elevada, otro por usarlo < 6 noches). Los 22 pacientes estudiados (3 mujeres): edad 61±11 años, IMC 32.2±5 kg/m2, circunferencia de cuello 44.5±4.7cm, IAH 45±20/h (n=18), Indice de desaturación de 4% 36±12/h (n=4). El APAP fue usado por 16 pacientes las 7 noches. El tiempo de uso promedio en 1 vs. 3 vs. 7 noches fue de 7.1±1.2 vs. 7.0±0.9 vs. 6.7±1.1 h/noche (p<0.05 al comparar 1 y 3 vs. 7 noches). La presión eficaz para abolir los eventos obstructivos durante el 95% del trazado (P95) fue de 9.6±2.6 vs. 9.9±2.3 vs. 9.8±2.3 cmH2O (p NS). Analizando en cada paciente las P95 obtenidas en los distintos períodos estudiados, encontramos diferencias menor o - =1 cmH2O en 6 pacientes entre la primera y el promedio de 7 noches (p<0.05), y en 1 paciente comparando 3 y 7 noches (p NS). Conclusión: El registro de 3 noches mostró resultados similares a los obtenidos con 7 noches de autotitulación. Por el contrario, una sola noche resultó insuficiente debido a significativa variabilidad.

Automatic devices for auto-titrating positive airway pressure (APAP) enable to estimate the effective pressure level of treatment in patients with sleep apnea, without supervision by a technician and at a lower cost than attended polysomnography titration. One of these devices, Autoset (ResMed), modifies the delivered pressure based on the presence of apneas and inspiratory flow limitation. Patients with sleep apnea-hypopnea syndrome were studied with this device during a week, after more than 60 days of continuous positive airway pressure (CPAP) with anarbitrary pressure between 8 and 9 cm H20. The study compared the pressure level measured in 1, 3 and 7 nights. The recordings were considered acceptable if at least 6 nights were included, and the mean leak was less than 0.4 l/second. Twenty four patients were recruited for the study; 2 were excluded (one by high leak level, and another by using the equipment < 6 nights). The 22 patients included in the analysis were 3 females and 19 males: age 61 ±11 years, BMI 32.2 ±5 kg/m2, neck circumference 44.5 ±4.7cm, Apnea Hypopnea Index (AHI) 45 ±20/h (n=18), oxygen desaturation index of 4% 36 ±12/h (n=4). The APAP was used across 7 nights by 16 patients. Mean time of use in 1 versus 3 versus 7 nights was respectively 7.1 ±1.2, 7.0 ±0.9 and 6.7 ±1.1 h/night (p < 0.05 comparing 1 and 3 versus 7 nights). Effective pressure to abolish obstructive events during 95% of the registered time (P95) was respectively 9.6 ±2.6, 9.9 ±2.3 and 9.8 ±2.3 cm water (pNS). Analyzing the individual P95 in the different periods, there was a difference of - > 1cm H20 in 6 patients between the first night and the mean of 7 nights (p < 0.05); in 1 patient this difference was observed comparing 3 and 7 nights (p NS). Conclusion: The registers of autotitrating during 3 nights and 7 nights were similar. On the other hand the observation of only one night was insufficient because of significant variability.