TUNEL Labeling Research Articles

Anti-oxidant, anti-apoptotic and anti-inflammatory effects of geraniin in spinal cord injury in rat: role of COX-2.

Spinal cord injury (SCI) is devastating diseases affecting the degeneration of the spinal column, and vascular problems. However, the currently available therapeutic interventions are insufficient to address the effect of SCI which leads to significant impact on the morbidity and mortality of patients. In the present manuscript, we intend to investigate the pharmacological effect of geraniin on the SCI in Sprague-Dawley (SD) rats. The SCI in rats were induced by the conventional weight-drop method and treated with GER (2.5, 5, and 10 mg/kg). Subsequently, the locomotor activity of rats with SCI was assessed using Basso, Beattie, and Bresnahan (BBB) scores, while oxidative stress indicators and inflammatory variables were analyzed using commercially available kits. Additionally, neuronal death was quantified using TUNEL labeling. The enzymatic activity of caspase 3, 8, and 9 was also assessed. Furthermore, the expression levels of Bcl2, Bax, and COX-2 in rat spinal cords after SCI were analyzed by RT-PCR analysis. Our research indicated that therapy with GER in a manner that depends on the dosage could enhance the functional recovery, as well as reduce the occurrence of apoptosis, mitigate the inflammatory and oxidative response in rats with SCI. Furthermore, it was observed that GER increased the expression of Bcl2 and decreased the expression of Bax and COX-2. The concentration of caspase-3, -8, and -9 was observed to be decreased in SCI rats treated with GER. GER might protect the spinal cord from SCI by reducing apoptosis, oxidative stress, and inflammatory response through the inhibition of COX-2.

Influence of Metamizole on Antitumour Activity of Risedronate Sodium in In Vitro Studies on Canine (D-17) and Human (U-2 OS) Osteosarcoma Cell Lines.

The availability of metamizole varies greatly around the world. There are countries such as the USA, UK, or Australia where the use of metamizole is completely forbidden, and there are also countries where this drug is available only on prescription (e.g., Greece, Italy, Spain, etc.) and those in which it is sold OTC-over the counter (e.g., most Asian and South American countries). Metamizole, as a drug with a strong analgesic effect, is used as an alternative to other non-steroidal anti-inflammatory drugs, alone or in combination with opioid drugs. Risedronate sodium is a third-generation bisphosphonate commonly used in orthopaedic and metabolic diseases of the musculoskeletal system, including hypercalcemia, postmenopausal osteoporosis, Paget's disease, etc. The aim of this study was to check whether there were any pharmacological interactions between metamizole and risedronate sodium in in vitro studies. Cell viability was assessed using the MTT method, the number of apoptotic cells was assessed using the labelling TUNEL method, and the cell cycle assessment was performed with a flow cytometer and propidium iodide. This was a pilot study, which is why only two cancer cell lines were tested: D-17 of canine osteosarcoma and U-2 OS of human osteosarcoma. Exposure of the canine osteosarcoma cell line to a combination of risedronate sodium (100 µg/mL) and metamizole (50, 5, and 0.5 µg/mL) resulted in the complete abolition of the cytoprotective activity of metamizole. In the human osteosarcoma cell line, the cytotoxic effect of risedronate sodium was entirely eliminated in the presence of 50 µg/mL of metamizole. The cytoprotective and anti-apoptotic effect of metamizole in combination with risedronate sodium in the tested human and canine osteosarcoma cell lines indicates an urgent need for further in vivo studies to confirm or disprove the potential dose-dependent undesirable effect of such a therapy.

The Different Therapeutic Effects of Traditional Chinese Medicine Shensong Yangxin Capsule and Salubrinal in High-intensity Exercise-induced Heart Failure in Rats with Acute Myocardial Infarction.

Currently, endoplasmic reticulum stress is studied utilizing a dephosphorylation inhibitor (Sal). The traditional Chinese patent medicine and simple formulation Shensong Yangxin Capsule is a commonly used medication for the treatment of arrhythmia. However, the efficacy and underlying mechanism of the capsule in treating post-ischemic heart failure in myocardial tissue have not yet been investigated. The therapeutic effects and the underlying mechanism of the Shensong Yangxin Capsule (SSYX) and the dephosphorylation inhibitor Salubrinal (Sal) on heart failure (HF) induced by high-intensity exercise in rats with acute myocardial infarction (AMI) were investigated. Male infants of 8 weeks Spragge-Dawley (SD) rats were randomly assigned to one of four groups: sham surgery group, AMI+placebo group, AMI+Shensong Yangxin Capsule group (AMI+SSYX), and AMI+Sal administration group. Rats' myocardial infarction was induced by left coronary artery ligation. Rats were subjected to a 3-week high-intensity exercise program to simulate heart failure after 7 days of postoperative rest. After the fourth postoperative week, echocardiography was applied to determine the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and left ventricular systolic volume (LVESV) in each group. HE and TUNEL labeling were employed to examine the morphology of cardiac cells and measure the percentage of apoptosis in each group; Western blotting was applied to detect the cardiomyocyte apoptosis-related proteins p-JNK, p-P38, and NOX2, while ELISA was used to detect glutathione(GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) in serum. Following a 4-week drug intervention:(1)LVFS and LVEF in the AMI+placebo group were statistically significantly reduced, while LVESV were significantly higher, compared to those in the sham surgery group (P<0.05); The AMI+SSYX group performed statistically significantly better than the AMI+placebo group(P<0.05). (2) The myocardial cells in the AMI+placebo group exhibited significant swelling and inflammatory cell infiltration; the myocardial cells in the AMI+SSYX group and AMI+Sal group displayed mild swelling and minimal inflammatory cell infiltration; the AMI+SSYX group's myocardial cell morphology was superior to that of the AMI+Sal group; (3) The apoptosis rate of the AMI+placebo group was around 95%, greater than that of the sham surgery group (2.55%). The apoptosis rate of the AMI+SSYX group is approximately 21%, while the apoptosis rate of the AMI+Sal group is about 43%. (4) In the AMI+placebo group, p-JNK, p-P38, and NOX2 protein expression dramatically increased compared to the sham surgery group. The expression of p-P38, NOX2, and p-JNK/t-JNK was considerably reduced in the AMI+Shensong group and AMI+Sal group, compared to the AMI+placebo group. (P<0.01)The AMI+SSYX group's result is superior to that of the AMI+Sal group. (5) Compared to the sham surgery group, the serum levels of SOD and GSH were significantly lower, and MDA was significantly higher in the AMI+placebo group. Compared to the AMI+placebo group, the serum levels of SOD and GSH were significantly higher, and MDA was significantly lower in the AMI+SSYX group and the AMI+Sal group. (P<0.05) Conclusion: In rats with acute myocardial infarction in high-intensity exercise-induced heart failure, Shensong Yangxin Capsule dramatically reduces myocardial cell death and cardiac dysfunction. SSYX has a shorter course of treatment and a better therapeutic effect than Sal.

LncRNA HILPDA promotes contrast-induced acute kidney injury by recruiting eIF4B to upregulate XPO1 expression.

Contrast-induced acute kidney injury (CI-AKI) is a serious and common complication following the use of iodinated contrast media, with a 20% fatality rate. The function of long non-coding RNA HILPDA (lnc-HILPDA) in CI-AKI development was investigated in this study. CI-AKI models were constructed by iopromide treatment. Kidney pathological changes were analyzed by HE staining. TUNEL labeling and flow cytometry were used to examine cell apoptosis. CCK-8 assay was used to determine cell viability. The interactions between lnc-HILPDA, eIF4B, and XPO1 were verified by RIP or Co-IP assay. Lnc-HILPDA was upregulated in CI-AKI, and its knockdown decreased contrast-trigged oxidative stress and apoptosis in HK-2 cells. Mechanically, lnc-HILPDA activated the NF-κB pathway by upregulating XPO1 through interacting with eIF4B. Moreover, the inhibitory effect of lnc-HILPDA downregulation on contrast-induced oxidative stress and apoptosis in HK-2 cells was weakened by XPO1 overexpression. Lnc-HILPDA accelerated CI-AKI progression by elevating XPO1 expression through eIF4B to activate NF-κB pathway.

Apoptosis of hippocampus and cerebellum induced with brain ischemia reperfusion prevented by 3’,4’-dihydroxyflavonol (DiOHF)

ABSTRACT The present study aimed to determine the effect of 3',4'-dihydroxyflavonol (DiOHF) on apoptosis in the cerebellum and hippocampus in rats with ischemia-reperfusion. A total of 38 Wistar albino male rats were used. Experimental groups were designed as Group 1-Sham; Group 2-Ischemia-reperfusion (IR), in which animals were anesthetized and carotid arteries ligated for 30 minutes (ischemia) and reperfused 30 minutes; Group 3- IR + DiOHF (10 mg/kg); Group 4- Ischemia + DiOHF (10 mg/kg) + reperfusion; Group 5-DiOHF + IR. DiOHF was supplemented as 10 mg/kg by intraperitoneal injection 30 minutes before IR. Following application, the animals were sacrificed under general anesthetic by cervical dislocation, and the cerebellum and hippocampus tissues were analyzed for apoptosis. IR significantly increased hippocampus and cerebellum apoptosis activity, confirmed by Hematoxylin-Eosin, TUNEL labeling, and Caspase-8 activity. However, these values were significantly suppressed by the administration of DiOHF, especially when used before the ischemia and reperfusion. The results of the study show that increased apoptosis in the cerebellum and hippocampus tissue was inhibited by intraperitoneal DiOHF supplementation.

Repeated boat noise exposure damages inner ear sensory hair cells and decreases hearing sensitivity in Atlantic croaker (Micropogonias undulatus).

Anthropogenic noise is becoming a major underwater pollutant because of rapidly increasing boat traffic worldwide. But its impact on aquatic organisms remains largely unknown. Previous studies have focused mainly on high-frequency and impulsive noises (i.e. sonar); however, boat noise is more pervasive, continuous, and its highest intensity and component frequencies overlap the auditory bandwidth of most fishes. We assessed the impacts of boat noise on saccular sensory hair cell density and hearing thresholds of a soniferous species, Atlantic croaker (Micropogonias undulatus). In two laboratory experiments, individuals were subjected to simulated boat noise: a single 15-min exposure and 3days of intermittent noise (simulating passing vessels). Immediately after both experiments, fish were either (1) tested for hearing sensitivity with auditory evoked potential (AEP) tests or (2) euthanized for fluorescent phalloidin and TUNEL labeling for hair cell density counts. Relative to controls, no differences were observed in auditory thresholds nor hair cell density between individuals subjected to a single 15-min noise exposure. However, fish from the 3-day experiment showed decreased sensory hair cell density, increased apoptotic cells, and higher hearing thresholds than control fish at 300, 800 and 1000 Hz. Our results demonstrate that impacts from boat noise depend upon the duration and frequency of exposure. For a species reliant on vocalization for communication, these impacts may hinder spawning success, increase predation risks and significantly alter the ecosystem.

Heat-shock protein 90 alleviates oxidative stress and reduces apoptosis in liver of Seriola aureovittata (yellowtail kingfish) under high-temperature stress

Hsp90s are molecular chaperones that enhance fish tolerance to high-temperature stress. However, the function of Hsp90s in Seriola aureovittata (yellowtail kingfish) under high-temperature stress remains largely unknown. Here, two Hsp90 isoforms were identified in S. aureovittata by bioinformatics analysis: SaHsp90α and SaHsp90β. The coding sequence of SaHsp90α was 2193-bp long and encoded a polypeptide of 730 amino acids; SaHsp90β was 2178-bp long and encoded a polypeptide of 725 amino acids. SaHsp90α and SaHsp90β both contained a HATPase domain and a HSP90 domain. Their transcripts were detected in all examined S. aureovittata tissues, with relatively high levels in the gonads, head kidney, and intestine. During high-temperature stress at 28 °C, the expression levels of SaHsp90α and SaHsp90β transcripts were significantly increased in liver. After simultaneously knocking down the expression of the SaHsp90s, there was a significant decrease in liver superoxide dismutase (SOD) activity and a remarkable increase of malondialdehyde content in liver after high-temperature stress. The expression levels of the key caspase family genes caspase-3 and caspase-7 were also significantly upregulated by high-temperature stress in SaHsp90-knockdown liver. TUNEL labeling demonstrated that the number of apoptotic cells significantly increased in the SaHsp90-knockdown group when high-temperature treatment lasted for 48 h. Protein–protein docking analysis predicted that SaHsp90α and SaHsp90β can bind to S. aureovittata SOD and survivin, which are key proteins for maintenance of redox homeostasis and inhibition of apoptosis. These findings demonstrate that SaHsp90α and SaHsp90β play a crucial role in resistance to high-temperature stress by regulating redox homeostasis and apoptosis in yellowtail kingfish.

Caprine arthritis encephalitis virus-induced apoptosis associated with brain lesions in naturally infected kids

Acute demyelinating leucoencephalomyelitis was the most conspicuous microscopic change in the brain and spinal cord of kids infected with caprine arthritis encephalitis virus (CAEV). TUNEL positivity and labelling of anti-bax and anti-caspases-3, -8 and -9 were found in a distinct population of glial cells, mainly at the edges of the demyelinated plaques and perivascular areas and, to a lesser extent, in neurons. Double labelling revealed that most of these apoptotic cells in the demyelinated plaques were astrocytes and a few were oligodendroglia. In contrast, expression of bcl-2, an anti-apoptotic protein, was found mainly in neurons of the brainstem and cerebellum and motor neurons of the spinal cord, but was restricted in glial cells. These results suggest that apoptosis plays an important role in the pathogenesis of CAE demyelinating encephalitis.

Evaluation of oxidative stress cycle in healthy and inflamed dental pulp tissue: a laboratory investigation.

The purpose of this study was to investigate the oxidative stress cycle consisting of reactive oxygen molecules (ROS), glutathione (GSH) and glutathione S-transferase (GST) in caries-related pulp inflammation. Fifty-four pulp tissue samples were collected from healthy donors with the diagnosis of reversible pulpitis, symptomatic irreversible pulpitis, and healthy pulp. Twelve pulp samples from each group were homogenized and total protein, ROS, GSH, and GST were measured by spectrophotometer. The remaining 6 samples from each group were prepared for paraffin block and used for the histopathologic and immunohistochemical evaluation of oxidative stress parameters and TUNEL labeling. Data were analyzed statistically. The results revealed that total protein levels significantly decreased; however, ROS levels increased in both reversible and irreversible pulpitis compared to the healthy pulp (p < 0.01). Also, as inflammation increases, GST enzyme levels decrease while GSH levels increase significantly (p < 0.05). It was found that the number of TUNEL (+) cells was increased in irreversible pulpitis samples compared to healthy and reversible pulpitis groups (p < 0.05). GSTP1 and GSH immunoreactivity were also observed in irreversible pulpitis samples. It has been revealed that caries-related inflammation alters the oxidative stress cycle in dental pulp tissue. The increase in GSH levels in the inflamed dental pulp due to the increase in ROS levels may improve the defensive ability of the dental pulp. There is a relationship between oxidative stress and inflammation. Control of excessive oxidative stress in pulpitis can stimulate reparative and regenerative processes. The present findings may provide an overview of the management of oxidative stress in cases with pulpitis during regenerative treatments.

Flow cytometric method for the detection and quantification of retinal cell death and oxidative stress

Retinal cell death is the major cause of vision loss in many forms of blinding retinal disease. A plethora of research is focused on understanding the mechanisms of retinal cell death to identify potential neuroprotective strategies that prevent vision loss in these diseases. Traditionally, histological techniques have been used to determine the type and extent of cell death in the retina. These techniques, such as TUNEL labeling and immunohistochemistry, are laborious and time consuming, resulting in low throughput and variable results depending on the experimenter. To increase throughput and reduce variability, we developed several flow cytometry-based assays to detect and quantify retinal cell death. The methods and accompanying data presented demonstrate that flow cytometry can readily detect both retinal cell death and oxidative stress and importantly, the efficacy of neuroprotective agents. These methods will be of interest to investigators looking to increase throughput and efficiency without compromising sensitivity as the methods herein reduce analysis time from several months to less than a week. As such, the flow cytometry methods presented have the potential to expedite research efforts focused on developing novel strategies for retinal cell neuroprotection.

Immediate effects of acute Mars mission equivalent doses of SEP and GCR radiation on the murine gastrointestinal system-protective effects of curcumin-loaded nanolipoprotein particles (cNLPs).

Missions beyond low Earth orbit (LEO) will expose astronauts to ionizing radiation (IR) in the form of solar energetic particles (SEP) and galactic cosmic rays (GCR) including high atomic number and energy (HZE) nuclei. The gastrointestinal (GI) system is documented to be highly radiosensitive with even relatively low dose IR exposures capable of inducing mucosal lesions and disrupting epithelial barrier function. IR is also an established risk factor for colorectal cancer (CRC) with several studies examining long-term GI effects of SEP/GCR exposure using tumor-prone APC mouse models. Studies of acute short-term effects of modeled space radiation exposures in wildtype mouse models are more limited and necessary to better define charged particle-induced GI pathologies and test novel medical countermeasures (MCMs) to promote astronaut safety. In this study, we performed ground-based studies where male and female C57BL/6J mice were exposed to γ-rays, 50 MeV protons, or 1 GeV/n Fe-56 ions at the NASA Space Radiation Laboratory (NSRL) with histology and immunohistochemistry endpoints measured in the first 24 h post-irradiation to define immediate SEP/GCR-induced GI alterations. Our data show that unlike matched γ-ray controls, acute exposures to protons and iron ions disrupts intestinal function and induces mucosal lesions, vascular congestion, epithelial barrier breakdown, and marked enlargement of mucosa-associated lymphoid tissue. We also measured kinetics of DNA double-strand break (DSB) repair using gamma-H2AX- specific antibodies and apoptosis via TUNEL labeling, noting the induction and disappearance of extranuclear cytoplasmic DNA marked by gamma-H2AX only in the charged particle-irradiated samples. We show that 18 h pre-treatment with curcumin-loaded nanolipoprotein particles (cNLPs) delivered via IV injection reduces DSB-associated foci levels and apoptosis and restore crypt villi lengths. These data improve our understanding of physiological alterations in the GI tract immediately following exposures to modeled space radiations and demonstrates effectiveness of a promising space radiation MCM.

Dexmedetomidine alleviates myocardial ischemia–reperfusion injury by down-regulating miR-34b-3p to activate the Jagged1/Notch signaling pathway

BackgroundMyocardial ischemia/reperfusion (I/R) injury is a fatal event that usually occurs after reperfusion therapy for myocardial infarction. Dexmedetomidine (Dex) has been shown to be beneficial in the treatment of myocardial infarction, however, its underlying mechanism for regulating I/R injury is unclear. MethodsH9c2 cell and rat models of I/R injury were established via oxygen-glucose deprivation reoxygenation (OGD/R) and occlusion of the left anterior descending branch of coronary artery, respectively. Flow cytometry, MTT, or DHE assay detected cell activity, ROS, or apoptosis, respectively. The expression levels of miR-34b-3p and related mRNAs were determined using qRT-PCR. Related protein expression levels were detected by Western blotting and ELISA test. The interaction between miR-34b-3p and Jagged1 was assessed by dual luciferase reporter and RIP assays. The morphology of cardiac tissue was examined by TTC, HE, and TUNEL labeling. ResultsDex markedly inhibited the inflammatory damage and apoptosis caused by OGD/R in H9c2 cells. MiR-34b-3p and Jagged1 levels were increased and decreased in myocardial I/R injury model, respectively, while Dex reversed this effect. Moreover, miR-34b-3p was firstly reported to directly bind and decrease Jagged1 expression, thereby inhibiting Notch signaling pathway. Transfection of agomiR-34b-3p or Jagged1 silencing eliminated Dex’s defensive impact on OGD/R-induced cardiomyocytes damage. Dex relieved the myocardial I/R injury of rats via inhibiting miR-34b-3p and further activating Notch signaling pathway. ConclusionDex protected myocardium from I/R injury via suppressing miR-34b-3p to activate Jagged1-mediated Notch signaling pathway. Our findings revealed a novel mechanism underlying of Dex on myocardial I/R injury.

ДИФФЕРЕНЦИРОВКА МИОБЛАСТОВ, ВЫДЕЛЕННЫХ ИЗ M. SOLEUS КРЫСЫ ПОСЛЕ АНТИОРТОСТАТИЧЕСКОГО ВЫВЕШИВАНИЯ, СОПРОВОЖДАЕТСЯ ДЕФОСФОРИЛИРОВАНИЕМ P27KIP1 И УСИЛЕНИЕМ АПОПТОЗА

Apoptosis and its control during differentiation of primary myoblasts isolated from rat soleus muscle after 7-d tail suspension were investigated. Primary myoblasts were first subjected to myogenic differentiation. TUNEL labeling of double-strand DNA breaks was applied to detect apoptotic cells during myoblast differentiation. Western blot was used to determine the apoptosis markers and a number of signaling molecules, i.e. protein BAX, activated caspase-3, phospho-AMPK (Thr172), phospho-AKT (Ser 437), phospho-p27Kip1 (Thr198), phospho-p27Kip1 (Thr157). PCR analysis was used to investigate the expression of pro-apoptotic markers in myoblasts. The investigation showed enhancement of apoptosis as well as АМРК and p27Kip1 dephosphorylation in the process of differentiation of myoblasts taken from m. soleus of tail-suspended rats.

Erxian decoction alleviates cisplatin-induced premature ovarian failure in rats by reducing oxidation levels in ovarian granulosa cells

Ethnopharmacological relevantErxian Decoction (EXD) has been used empirically for more than 70 years to treat premature ovarian failure (POF), but more research is needed to understand how it works. Aim of the researchThe study aims to ascertain both in vivo and in vitro rewards of EXD. Materials and methodsEXD is composed of Curculiginis Rhizoma, Epimedii Folium, Morindae Officinalis, Angelicae Sinensis, Anemarrhenae Rhizoma, and Phellodendri Chinensis Cortex. UPLC/MS analysis was used to investigate the components of EXD. Using a POF model created by administering cisplatin to rats intraperitoneally, the pharmacodynamic effects of EXD were investigated. Three dose groups of EXD were garaged into rats: high (15.6 g/kg), medium (7.8 g/kg), and low (3.9 g/kg). By using a vaginal smear, the impact of EXD on the rat estrous cycle was evaluated. An ELISA test was used to measure the anti-Mullerian hormone (AMH), estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels in the serum of rats. By using HE stains, pathological alterations in the ovaries may be seen. MDA and SOD levels in ovarian samples were used to measure the degree of ovarian oxidation. TUNEL labeling of ovarian sections was used to find apoptosis levels. By using ATP, energy production was evaluated. The relative expression of proteins connected to aging and the RAGE pathway was assessed using Western blot. Then, using H2O2, a model of senescent human ovarian granulosa cells (KGN) was created in vitro. The impact of EXD and H2O2 on cellular senescence was discovered using-galactosidase staining. Cell apoptosis levels were found using PI/Hoechest33342. By using DCFH-DA, intracellular ROS was examined. MDA and SOD concentrations were used to measure the degree of cellular oxidation. RAGE-related mRNA and protein expression were evaluated using RT-qPCR and western blotting. ResultsUsing UPLC/MS analysis, 39 chemicals in EXD were found. Rats' estrous cycles were enhanced by EXD, which increased ovarian index and follicle count and reduced the proportion of atretic follicles in the rats. EXD reduced LH and FSH output while restoring AMH and E2 secretion. In ovarian tissues, EXD reduced the amount of apoptosis and MDA while raising SOD activity and ATP levels. The protein levels of p16, p21, p53, and Lamin A/C were among the senescence-related proteins that EXD lowered, along with the levels of RAGE, PI3K, BAX, and CASPASE 3. Anti-apoptotic protein BCL-2 was also raised in the RAGE pathway. Senescence, apoptosis, ROS, and MDA levels in the KGN cells were lowered in vitro by EXD. Additionally, EXD increased the anti-apoptotic potential by changing the expression of CAT, SOD2, and SIRT1. RAGE, BAX, BCL-2, CASPASE 3, and p38 expression levels were altered by EXD, enhancing its anti-apoptotic capability. ConclusionEXD boosted the ovary's antioxidant and anti-apoptotic capabilities while enhancing the estrous cycle and hormone output. These findings strongly suggested that EXD may contribute to the alleviation of POF and ovarian granulosa cells senescence.

Long-Term Consumption of Food-Derived Chlorogenic Acid Protects Mice against Acetaminophen-Induced Hepatotoxicity via Promoting PINK1-Dependent Mitophagy and Inhibiting Apoptosis.

Hepatotoxicity brought on by acetaminophen (APAP) is significantly impacted by mitochondrial dysfunction. Mitophagy, particularly PINK1-mediated mitophagy, maintains the stability of cell function by eliminating damaged mitochondria. One of the most prevalent dietary polyphenols, chlorogenic acid (CGA), has been shown to have hepatoprotective properties. It is yet unknown, nevertheless, whether its defense against hepatocyte apoptosis involves triggering PINK1-mediated mitophagy. In vitro and in vivo models of APAP-induced hepatotoxicity were established to observe CGA's effect and mechanism in preventing hepatotoxicity in the present study. Serum aminotransferase levels, mouse liver histology, and the survival rate of HepG2 cells and mice were also assessed. The outcomes showed that CGA could reduce the activities of serum enzymes such as alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH), and alleviate liver injury in mice. It could also significantly increase the cell viability of HepG2 cells and the 24-h survival rate of mice. TUNEL labeling and Western blotting were used to identify the hepatocyte apoptosis level. According to data, CGA could significantly reduce liver cell apoptosis in vivo. Additionally, Tom20 and LC3II colocalization in mitochondria may be facilitated by CGA. CGA considerably increased the levels of genes and proteins associated with mitophagy (PINK1, Parkin, LC3II/LC3I), while considerably decreasing the levels of p62 and Tom20, suggesting that it might activate PINK1/Parkin-mediated mitophagy in APAP-induced liver damage. Additionally, the protection of CGA was reduced when PINK1 was knocked down by siPINK1 in HepG2 cells, and it did not upregulate mitophagy-related proteins (PINK1, Parkin, LC3II/LC3I). In conclusion, our findings revealed that long-term consumption of food-derived CGA could prevent APAP hepatotoxicity via increasing PINK1-dependent mitophagy and inhibiting hepatocyte apoptosis.

Alterations in hippocampal neurogenesis and hippocampal insulin signaling pathway in rat with metabolic syndrome.

We aimed to examine the level of hippocampal neurogenesis, and assess learning and anxiety and the level of some proteins involving insulin signaling pathways in rats with Metabolic Syndrome (MetS); and to reveal the relationship among them. Totally, 30 Wistar-albino rats were used. The rats were divided into three groups: Control, MetS, and MetS+Ins. Immunohistochemical staining was performed to evaluate the levels of neurogenesis markers; Doublecortin (DCX), Neuronal-Differentiation-1 (NeuroD1), Ki67, and Neuronal nuclear protein (NeuN). Then, cleaved caspase-3 and TUNEL labeling were performed to detect the level of apoptosis. Additionally, behavior tests were performed to evaluate the learning-memory levels and anxiety-like behaviors. Insulin, Insulin Receptor (IR), Insulin Receptor Substrate (IRS2), glucose transporter (GLUT)-3, and GLUT4 protein expression levels were analyzed to evaluate the possible changes in the insulin signaling pathway. An increase in anxiety with memory deficiency was observed in MetS. In the hippocampus of MetS, an increase was detected in the level of apoptosis, whereas a decrease was detected in the expression level of the neurogenesis marker. Insulin secretion and IR levels decreased in hippocampal neurons. We observed that GLUT3 and GLUT4 levels increased because of the non-activated insulin signaling pathway. We think that the insulin signaling pathway may have an effect on the decreased neurogenesis in the MetS group. So, the evaluation of the Mitogen-activated protein kinase (MAPK) pathway and the investigation of the effect of endoplasmic reticulum stress on this pathway will be among the targets of our future studies.

Putative COVID-19 therapies imatinib, lopinavir, ritonavir, and ivermectin cause hair cell damage: A targeted screen in the zebrafish lateral line.

The biomedical community is rapidly developing COVID-19 drugs to bring much-need therapies to market, with over 900 drugs and drug combinations currently in clinical trials. While this pace of drug development is necessary, the risk of producing therapies with significant side-effects is also increased. One likely side-effect of some COVID-19 drugs is hearing loss, yet hearing is not assessed during preclinical development or clinical trials. We used the zebrafish lateral line, an established model for drug-induced sensory hair cell damage, to assess the ototoxic potential of seven drugs in clinical trials for treatment of COVID-19. We found that ivermectin, lopinavir, imatinib, and ritonavir were significantly toxic to lateral line hair cells. By contrast, the approved COVID-19 therapies dexamethasone and remdesivir did not cause damage. We also did not observe damage from the antibiotic azithromycin. Neither lopinavir nor ritonavir altered the number of pre-synaptic ribbons per surviving hair cell, while there was an increase in ribbons following imatinib or ivermectin exposure. Damage from lopinavir, imatinib, and ivermectin was specific to hair cells, with no overall cytotoxicity noted following TUNEL labeling. Ritonavir may be generally cytotoxic, as determined by an increase in the number of TUNEL-positive non-hair cells following ritonavir exposure. Pharmacological inhibition of the mechanotransduction (MET) channel attenuated damage caused by lopinavir and ritonavir but did not alter imatinib or ivermectin toxicity. These results suggest that lopinavir and ritonavir may enter hair cells through the MET channel, similar to known ototoxins such as aminoglycoside antibiotics. Finally, we asked if ivermectin was ototoxic to rats in vivo. While ivermectin is not recommended by the FDA for treating COVID-19, many people have chosen to take ivermectin without a doctor’s guidance, often with serious side-effects. Rats received daily subcutaneous injections for 10 days with a clinically relevant ivermectin dose (0.2 mg/kg). In contrast to our zebrafish assays, ivermectin did not cause ototoxicity in rats. Our research suggests that some drugs in clinical trials for COVID-19 may be ototoxic. This work can help identify drugs with the fewest side-effects and determine which therapies warrant audiometric monitoring.

Aristolochic Acid Nephropathy: A Novel Suppression Strategy of Carbon Dots Derived from Astragali Radix Carbonisata

Despite strict restrictions on the use of aristolochic acids (AAs)-containing merchandise or drugs in many countries, a substantial amounts of occurrences aristolochic acid nephropathy (AAN) had been accounted worldwide. Clinically, there is no effective incurable therapy regimen to reverse the progression of AAN. Although carbon dots have shown surprising bioactivity, research on the acute kidney injury caused by AAs is lacking. Here, a novel biomass-carbon dots from Astragali Radix (AR) as precursors was synthesized through one-step pyrolysis treatment. The ARC-carbon dots (ARC-CDs) was demonstrated in detail for its inhibitory effect on aristolochic acid nephropathy in a mice model. The indexes of inflammatory cytokines as well as oxidative stress were significantly reduced by the ARC-CDs in kidney tissue cells. Additionally, the ARC-CDs administration resulted in a large decrease in positive apoptotic cells according to TUNEL labeling and western blotting, which may be connected to the ARC-CDs’ modulation of the protein in the Akt/Mdm2/p53 signaling pathway. These findings show that ARC-CDs have remarkable anti-inflammatory, antioxidant, and anti-apoptotic capabilities against acute kidney injury spurred by aristolochic acids via the AKT/Mdm2/p53 signaling pathway.

Protective Effects of Vitamin C against Neomycin-Induced Apoptosis in HEI-OC1 Auditory Cell.

Ototoxic hearing loss results from hair cell death via reactive oxygen species (ROS) overproduction and consequent apoptosis. We investigated the effects of vitamin C (VC) on neomycin-induced HEI-OC1 cell damage, as well as the mechanism of inhibition. HEI-OC1 cells were treated with neomycin or with vitamin C (VC). The results indicated that VC had a protective effect on neomycin-induced HEI-OC1 cell death. Mechanistically, VC decreased neomycin-induced ROS generation, suppressed cell death, and increased cell viability. VC inhibited neomycin-induced apoptosis, ameliorated neomycin reduced antiapoptotic Bcl-2 expression, and suppressed neomycin increased expression of proapoptotic Bax, caspase-3 cleavage and caspase-8. TUNEL labeling demonstrated that VC blocked neomycin-induced apoptosis. Further study revealed that the effect of VC on neomycin-induced hair cell death was through interference with JNK activation and p38 phosphorylation. These results indicate that VC via suppressed ROS generation, which inhibited cell death by counteracting apoptotic signaling induced by neomycin in cells. Hence, VC is a potential candidate for protection agent against neomycin-induced HEI-OC1 cell ototoxicity.

Adipocyte GPX4 protects against inflammation, hepatic insulin resistance and metabolic dysregulation.

Metabolic inflammation is a hallmark of obesity and related disorders, afflicting substantial morbidity and mortality to individuals worldwide. White visceral and subcutaneous adipose tissue not only serves as energy storage but also controls metabolism. Adipose tissue inflammation, commonly observed in human obesity, is considered a critical driver of metabolic perturbation while molecular hubs are poorly explored. Metabolic stress evoked by e.g. long-chain fatty acids leads to oxidative perturbation of adipocytes and production of inflammatory cytokines, fuelling macrophage infiltration and systemic low-grade inflammation. Glutathione peroxidase 4 (GPX4) protects against lipid peroxidation, accumulation of oxygen-specific epitopes and cell death, collectively referred to as ferroptosis. Here, we explore the function of adipocyte GPX4 in mammalian metabolism. We studied the regulation and function of GPX4 in differentiated mouse adipocytes derived from 3T3-L1 fibroblasts. We generated two conditional adipocyte-specific Gpx4 knockout mice by crossing Gpx4fl/fl mice with Adipoq-Cre+ (Gpx4-/-AT) or Fabp4-Cre+ (Gpx4+/-Fabp4) mice. Both models were metabolically characterized by a glucose tolerance test and insulin resistance test, and adipose tissue lipid peroxidation, inflammation and cell death were assessed by quantifying oxygen-specific epitopes, transcriptional analysis of chemokines, quantification of F4/80+ macrophages and TUNEL labelling. GPX4 expression was induced during and required for adipocyte differentiation. In mature adipocytes, impaired GPX4 activity spontaneously evoked lipid peroxidation and expression of inflammatory cytokines such as TNF-α, interleukin 1β (IL-1β), IL-6 and the IL-8 homologue CXCL1. Gpx4-/-AT mice spontaneously displayed adipocyte hypertrophy on a chow diet, which was paralleled by the accumulation of oxygen-specific epitopes and macrophage infiltration in adipose tissue. Furthermore, Gpx4-/-AT mice spontaneously developed glucose intolerance, hepatic insulin resistance and systemic low-grade inflammation, when compared to wildtype littermates, which was similarly recapitulated in Gpx4+/-Fabp4 mice. Gpx4-/-AT mice exhibited no signs of adipocyte death. Adipocyte GPX4 protects against spontaneous metabolic dysregulation and systemic low-grade inflammation independent from ferroptosis, which could be therapeutically exploited in the future.