Sesamol Supplementation Mitigates Isoproterenol-Induced Cardiac Toxicity in Rats by Stabilizing Cardiac Mitochondrial and Lysosomal Enzymes

The current investigation was intended to evaluate the antimyocardial ischemic effects of sesamol on lactate dehydrogenase (LDH) isoenzymes, DNA damage, and mitochondrial and lysosomal enzyme activities in isoproterenol (ISO)-induced myocardial infarction (MI) in male albino Wistar strain rats. Rats that received ISO (85 mg/kg body weight (B.W) subcutaneously) for the first 2 consecutive days showed significant reduction in the activities of tricarboxylic acid (TCA) cycle enzymes (isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and succinate dehydrogenase) and respiratory chain enzymes (cytochrome c oxidase and nicotinamide adenine dinucleotide hydrogen (NADH) dehydrogenase) in the heart mitochondria. The activities of the lysosomal enzymes (α-and β-glucosidases, α and β-galactosidases, β-glucuronidase and β-N-acetyl glucosaminidase and cathepsin-B and cathepsin-D) were increased significantly in the heart homogenate of ISO-induced MI rats. ISO injection also increased the % of tail DNA, tail length, and tail moment and decreased the % of head DNA. Pretreatment with sesamol (50 mg/kg B.W) every day for a period of 9 days prevented the above abnormalities induced by ISO. In conclusion, it can be inferred that administration of sesamol has a potent beneficial role against ISO-induced damage to the mitochondria, lysosomes, and DNA, thereby preventing MI.

A Decade of Mighty Lipophagy: What We Know and What Facts We Need to Know?

Lipids are integral cellular components that act as substrates for energy provision, signaling molecules, and essential constituents of biological membranes along with a variety of other biological functions. Despite their significance, lipid accumulation may result in lipotoxicity, impair autophagy, and lysosomal function that may lead to certain diseases and metabolic syndromes like obesity and even cell death. Therefore, these lipids are continuously recycled and redistributed by the process of selective autophagy specifically termed as lipophagy. This selective form of autophagy employs lysosomes for the maintenance of cellular lipid homeostasis. In this review, we have reviewed the current literature about how lipid droplets (LDs) are recruited towards lysosomes, cross-talk between a variety of autophagy receptors present on LD surface and lysosomes, and lipid hydrolysis by lysosomal enzymes. In addition to it, we have tried to answer most of the possible questions related to lipophagy regulation at different levels. Moreover, in the last part of this review, we have discussed some of the pathological states due to the accumulation of these LDs and their possible treatments under the light of currently available findings.

Antioxidant and anti-inflammatory properties of the methanolic extract of Siphonochilus aethiopicus rhizomes

Introduction: Siphonochilus aethiopicus is a medicinal plant widely used in the treatment of many inflammatory conditions such as arthritis. The objective of this study was therefore to evaluate the antioxidant and anti-inflammatory properties of methanolic extract of S. aethiopicus rhizomes. Methods: The total phenolic compounds, flavonoid, and tannin content, as well as the in vitro antioxidant activity of the extract, were estimated. The in vivo anti-inflammatory activity was then evaluated in male mice aged 3 to 4 months using the arthritic mouse model induced by carrageenan (0.05 ml; 1%) and monosodium urate (MSU) crystals (26.6 mg/mL). Mice were treated with the methanolic extract of S. aethiopicus (75, 150, 300 mg/kg) and the reference drugs: indomethacin (3 mg/kg) and colchicine (1 mg/kg). The serum, splenic, and hepatic lysosomal enzymes were determined, and oxidative stress biomarkers were estimated. Paws were sectioned for histological analysis. Results: Results showed that S. aethiopicus extract had non-negligible concentrations of polyphenols, flavonoids, and tannins, which could confer it an antioxidant effect. Further, the methanolic extract of S. aethiopicus at different doses significantly (P < 0.05) reduced paw swelling, attenuated joint inflammation, limited the release of lysosomal enzymes, and improved antioxidant enzymes. Conclusion: The methanolic extract of S. aethiopicus has anti-inflammatory and antioxidant properties, and can be used to treat acute forms of gouty arthritis.

The Effect of Submaximal Exercise Followed by Short-Term Cold-Water Immersion on the Inflammatory State in Healthy Recreational Athletes: A Cross-Over Study

Cold-water immersion (CWI) after exercise is a method used by sportsmen to improve recovery. The aim of the study was to assess the effect of a 3 min CWI on the inflammatory state by measuring levels of interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor α (TNF-α), and transforming growth factor β1 (TGF-β1), and activities of α1-antitrypsin (AAT) and lysosomal enzymes, including arylsulfatase (ASA), acid phosphatase (AcP), and cathepsin D (CTS D), in the blood of healthy recreational athletes. Male volunteers (n = 22, age 25 ± 4.8 yr) performed a 30 min submaximal aerobic exercise, followed by a 20 min rest at room temperature (RT-REST) or a 20 min rest at room temperature with an initial 3 min 8 °C water bath (CWI-REST). Blood samples were taken at baseline, immediately after exercise, and after 20 min of recovery. The IL-6, IL-10, and TNF-α levels and the AAT activity increased significantly immediately after exercise. The IL-6 level was significantly higher after CWI-REST than after RT-REST. No changes in the activities of the lysosomal enzymes were observed. The effect of a 3 min CWI on the level of inflammatory markers during post-exercise recovery was limited. Thus, it might be considered as a widely available method of regeneration for recreational athletes.

The expression of prosaposin and its receptors, GRP37 and GPR37L1, are increased in the developing dorsal root ganglion

Prosaposin (PSAP), a highly conserved glycoprotein, is a precursor of saposins A–D. Accumulating evidence suggests that PSAP is a neurotrophic factor, as well as a regulator of lysosomal enzymes. Recently, the orphan G-protein-coupled receptors GPR37 and GPR37L1 were recognized as PSAP receptors, but their functions have not yet been clarified. In this study, we examined the distribution of PSAP and its receptors in the dorsal root ganglion (DRG) during development using specific antibodies, and showed that PSAP accumulates primarily in lysosomes and is dispersed throughout the cytoplasm of satellite cells. Later, PSAP colocalized with two receptors in satellite cells, and formed a characteristic ring shape approximately 8 weeks after birth, during a period of rapid DRG development. This ring shape, which was only observed around larger neurons, is evidence that several satellite cells are synchronously activated. We found that sortilin, a transporter of a wide variety of intracellular proteins containing PSAP, is strongly localized to the inner side of satellite cells, which contact the neuronal surface. These findings suggest that PSAP and GPR37/GPR37L1 play a role in activating both satellite and nerve cells.

Diagnosis of Mucopolysaccharidoses and Mucolipidosis by Assaying Multiplex Enzymes and Glycosaminoglycans

Mucopolysaccharidoses (MPS) and mucolipidosis (ML II/III) are a group of lysosomal storage disorders (LSDs) that occur due to a dysfunction of the lysosomal hydrolases responsible for the catabolism of glycosaminoglycans (GAGs). However, ML is caused by a deficiency of the enzyme uridine-diphosphate N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase, EC2.7.8.17), which tags lysosomal enzymes with a mannose 6-phosphate (M6P) marker for transport to the lysosome. A timely diagnosis of MPS and ML can lead to appropriate therapeutic options for patients. To improve the accuracy of diagnosis for MPS and ML in a high-risk population, we propose a combination method based on known biomarkers, enzyme activities, and specific GAGs. We measured five lysosomal enzymes (α-L-iduronidase (MPS I), iduronate-2-sulfatase (MPS II), α-N-acetylglucosaminidase (MPS IIIB), N-acetylglucosamine-6-sulfatase (MPS IVA), and N-acetylglucosamine-4-sulfatase (MPS VI)) and five GAGs (two kinds of heparan sulfate (HS), dermatan sulfate (DS), and two kinds of keratan sulfate (KS)) in dried blood samples (DBS) to diagnose suspected MPS patients by five-plex enzyme and simultaneous five GAGs assays. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) for both assays. These combined assays were tested for 43 patients with suspected MPS and 103 normal control subjects. We diagnosed two MPS I, thirteen MPS II, one MPS IIIB, three MPS IVA, two MPS VI, and six ML patients with this combined method, where enzymes, GAGs, and clinical manifestations were compatible. The remaining 16 patients were not diagnosed with MPS or ML. The five-plex enzyme assay successfully identified MPS patients from controls. Patients with MPS I, MPS II, and MPS IIIB had significantly elevated HS and DS levels in DBS. Compared to age-matched controls, patients with ML and MPS had significantly elevated mono-sulfated KS and di-sulfated KS levels. The results indicated that the combination method could distinguish these affected patients with MPS or ML from healthy controls. Overall, this study has shown that this combined method is effective and can be implemented in larger populations, including newborn screening.

Exploring cardioprotective potential of esculetin against isoproterenol induced myocardial toxicity in rats: in vivo and in vitro evidence

Background Esculetin is a natural coumarin derivative from various plants with multiple pharmacological effects. Hence, the present study was undertaken to explore the cardio protective potential of esculetin against isoproterenol induced myocardial toxicity in rats. Methods The treatment schedule was fixed for 28 days and the rats were divided into five groups of six each. Rats of group I received the normal saline and served as normal control, group II was received ISO (100 mg/kg body weight) for last two consecutive days of the study and served as disease control. Groups III and IV received esculetin 10 and 20 mg/kg body weight respectively once a day per oral for 28 days along with ISO for last two consecutive days of the study. Cardiac biomarkers such as CK-MB and LDH, membrane bound Na+ /K+ ATPases activity, myocardial lysosomal enzymes activity and tissue antioxidants status were estimated in the heart tissue samples. The histopathological changes in the myocardium were also assessed. Further, DPPH assay was done to evaluate the free radicals scavenging potential of esculetin. Cytoxicity assay, intracellular ROS levels by DCFDA assay and m-RNA expression of TNF-α, IL-6 and NF-κB by quantitative RT-PCR in H9c2 cell lines. Results The increased levels of CK-MB, LDH, LPO, myocardial lysosomal enzymes and membrane bound Na+ /K+ ATPase levels by ISO administration was significantly increased with concomitant decrease in tissue antioxidant enzymes such as GSH, Catalase, and SOD. Pre-treatment with esculetin for 28 days has significantly decreased the levels of cardiac bio-markers, lysosomal enzymes, membrane bound Na+ /K+ ATPase levels as well as Lipid peroxides which is in contrary to the ISO group. Amelioration of the antioxidant levels were also found in esculetin treated groups. Histopathological examination of heart reveals that myocardial degeneration, mononuclear cell infiltration was noticed in ISO treated rats, whereas the same was restored with esculetin treatment. In H9C2 cell lines esculetin could effectively reduced intracellular ROS inhibition and m-RNA expression of pro-inflammatory cytokines including TNF-α, IL-6 and NF-κB to prevent apoptosis or cell necrosis. Conclusion The study provides the evidence of cardioprotective potentials of esculetin against isoproterenol induced myocardial infarction by antioxidant and myocardial membrane stabilization along with in vitro protection from arsenic induced ROS cell necrosis or apoptosis in H9C2 cells.

Evaluation of artificial signal peptides for secretion of two lysosomal enzymes in CHO cells

Enzyme replacement therapy (ERT) is a scientifically rational and clinically proven treatment for lysosomal storage diseases. Most enzymes used for ERT are purified from the culture supernatant of mammalian cells. However, it is challenging to purify lysosomal enzymes with sufficient quality and quantity for clinical use due to their low secretion levels in mammalian cell systems. To improve the secretion efficiency of recombinant lysosomal enzymes, we evaluated the impact of artificial signal peptides on the production of recombinant lysosomal enzymes in Chinese Hamster Ovary (CHO) cell lines. We engineered two recombinant human lysosomal enzymes, N-acetyl-a-glucosaminidase (rhNAGLU) and glucosamine (N-acetyl)-6-sulfatase (rhGNS), by replacing their native signal peptides with 9 different signal peptides derived from highly secretory proteins and expressed them in CHO K1 cells. When comparing the native signal peptides, we found that rhGNS was secreted into media at higher levels than rhNAGLU. The secretion of rhNAGLU and rhGNS can, however, be carefully controlled by altering signal peptides. The secretion of rhNAGLU was relatively higher with murine Igk light chain and human chymotrypsinogen B1 signal peptides, whereas Igk light chain signal peptide 1 and human chymotrypsinogen B1 signal peptides were more effective for rhGNS secretion, suggesting that human chymotrypsinogen B1 signal peptide is the most appropriate for increasing lysosomal enzyme secretion. Collectively, our results indicate that altering signal peptide can modulate the secretion of recombinant lysosome enzymes and will enable lysosomal enzyme production for clinical use.

Levels of marker lysosomal hydrolases in women with coronary heart disease depending on age and sex hormone level

Aim of the study was to evaluate the serum concentrations of three marker lysosomal hydrolases (cathepsin D, acid phosphatase (AP) and acid DNase (aDNAase)) in women with coronary heart disease (CHD) depending on the level of follicle-­stimulating hormone (FSH), testosterone (T), age and find if those parameters associated with anthropometric parameters, glycemia, insulinemia and HOMA-IR index, biomarkers of atherosclerosis. The study included 285 women aged 35–65 years (median age was 54.4 years (25% and 75% percentiles — 43.2 and 61.3 years, respectively) who had had myocardial infarction no earlier than 30 days before the examination. Patients were divided into the following age groups: 35–55 and 56–65 years (first and second age groups, respectively), and into groups according to the levels of sex hormones: FSH ≥ and <30 mIU/mL and testosterone ≥ and <3 nmol/L. Results of comparative and correlation analyzes demonstrates that in women 35–65 years old with FSH ≥30 mIU/mL, the levels of cathepsin D are higher (p<0.05) than in patients with FSH <30 mIU/mL, and in women 35–55 years old, the content of AP was also higher (p=0.025). Associations of a high level of androgen with lysosomal hyperenzymemia were demonstrated only in the second age group, where at a level of T ≥3 nmol/L, higher values of all three lysosomal enzymes were recorded. Multivariate analysis in both age groups is confirmed direct impact of periand postmenopausal periods on the levels of lysosomal enzymemia and, accordingly, a negative effect on the state of lysosomal membranes. Thus, FSH levels directly determined the concentrations of AP and cardiotropic cathepsin D. The levels of aDNAase in women with CHD of 56–65 years of age were positively correlated with indicators that determine insulin-­glucose homeostasis: glycemia (p<0.001), HOMA-IR index (p<0.001). Such associations of three marker lysosomal enzymes demonstrate the primary contribution of FSH ≥30 mIU/mL to an increase in the concentration of lysosomal hydrolases in women with CHD35–65 years old and the correlation of aDNAase with the processes triggered by insulin resistance.