Xanthohumol-Induced Rat Glioma C6 Cells Death by Triggering Mitochondrial Stress

AIM: To investigate the underlying mechanisms of xanthohumol (XN) on the proliferation inhibition and death of C6 glioma cells. METHODS: To determine the effects of XN on C6 cells, cell proliferation and mortality after XN treatment were assessed by SRB assay and trypan blue assay respectively. Apoptotic rates were evaluated by flowcytometry after Annexin V-FITC/PI double staining. The influence of XN on the activity of caspase-3 was determined by Western blot (WB); and nuclear transposition of apoptosis-inducing factor (AIF) was tested by immunocytochemistry and WB. By MitoSOXTM staining, the mitochondrial ROS were detected. Mitochondrial function was also tested by MTT assay (content of succinic dehydrogenase), flow cytometry (mitochondrial membrane potential (MMP)—JC-1 staining; mitochondrial abundance—mito-Tracker green), immunofluorescence (MMP—JC-1 staining; mitochondrial morphology—mito-Tracker green), WB (mitochondrial fusion-fission protein—OPA1, mfn2, and DRP1; mitophagy-related proteins—Pink1, Parkin, LC3B, and P62), and high-performance liquid chromatography (HPLC) (energy charge). Finally, mitochondrial protein homeostasis of C6 cells after XN treatment with and without LONP1 inhibitor bortezomib was investigated by trypan blue assay (proliferative activity and mortality) and WB (mitochondrial protease LONP1). All cell morphology images were taken by a Leica Microsystems microscope. RESULTS: XN could lead to proliferation inhibition and death of C6 cells in a time- and dose-dependent manner and induce apoptosis of C6 cells through the AIF pathway. After long incubation of XN, mitochondria of C6 cells were seriously impaired, and mitochondria had a diffuse morphology and mitochondrial ROS were increased. The content of succinic dehydrogenase per cell was significantly decreased after XN insults of 24, 48, and 72 h. The energy charge was weakened after XN insult of 24 h. Furthermore, the MMP and mitochondrial abundance were significantly decreased; the protein expression levels of OPA1, mfn2, and DRP1 were down-regulated; and the protein expression levels of Pink1, Parkin, LC3B-II/LC3B-I, and p62 were up-regulated in long XN incubation times (24, 48, and 72 h). XN incubation with bortezomib for 48 h resulted in lower proliferative activity and higher mortality of C6 cells and caused the cell to have visible vacuoles. Moreover, the protein expression levels of LONP1 was up-regulated gradually as XN treatment time increased. CONCLUSION: These data supported that XN could induce AIF pathway apoptosis of the rat glioma C6 cells by affecting the mitochondria.

Histoarchitectural and Histoenzymatic Studies on Gizzard of Guinea Fowl (Numida meleagris)

Background: The gizzard is a muscular stomach of the Gastrointestinal tract of bird that holds sparing crystals which aids as the mechanical multure component for food material to be ingested by birds. Its specialty is to grinding of ingested food material after secretion of HCL (Hydrochloric Acid) and pepsinogen enzymes in the proventriculus. Methods: The twelve samples of gizzard of guinea fowl were fixed in 10% NBF (Neutral buffered formalin) and Bouin’s fluid for histological while chilled acetone and chilled ethanol for histochemical studies. The fresh unfixed tissues were used for the cryostat section for the demonstration of different enzymes.Result: The tubular glands were main feature of mucosal layer but sometime acinar glands also found as the replacement of tubular glands with lymphoid aggregation. The horizontal koilin was also noticed between long mucosal folds. Tunica submucosa was discovered in squeezed manner in comparison to the mucosa. The tunica muscularis has shown inner circular and outer longitudinal as the thick layer. It was intermingling with collagen and elastic fibers. Histochemically the gizzard was examined for glycogen, alkaline phosphatase, acid phosphatase and succinic dehydrogenase.

Macroscopic, Histomorphological and Histochemical Studies on the Kidneys of Guinea Fowl (Numida meleagris)

Background: Kidneys, the paired organ which essentially plays for excreting nitrogenous wastes, excessive water, inorganic salts and toxic substances produced during the process of body metabolism. Maintenance of osmotic regulation and homeostatic fluid balance of the body has also be performed by kidneys. Each of the kidneys filtrate carried to cloaca by respective ureter from where the urine leaves the body. The present macroscopic, histomorphological and histochemical studies undertaken on the Kidneys of Guinea fowl (Numida meleagris) is nothing but the attempt to widen our thrust area for that bird which is nonexistent as on documentation.Methods: The present studies were carried upon 12 pairs of in-vitro kidney samples in Guinea fowl. Gross morphology and morphometrical observations were carried on fresh specimens. Histological studies were carried on fixed samples those were processed for dehydration, clearing, embedding into paraffin and sections of 5-6 μ thickness were obtained. Different staining procedure as to Haematoxylin and Eosin, Silver orcein, Aniline blue and Weigert’s staining methods were employed for different histological components. The histochemical and histoenzymic studies were also accomplished with Periodic acid Schiff‘s (PAS), Alcian blue methods (pH-2.5) and Gomori’s cobalt method for Alkaline phosphatase and Acid phosphatase. The Succinic dehydrogenase enzyme was also estimated on cryostat sections.Result: The observations on kidneys of Guinea fowl were accomplished after macroscopic, histomorphological and histochemical means. This reddish-brown colored paired organ of kidneys was retroperitoneally placed in synsacral fossa and had incomplete division of three parts. The wrapping material of capsule was the dense connective tissue. The outer cortex and inner medulla had different parts of nephrons. Two types of nephrons viz. mammalian type (with Henle loop) and reptilian type (without Henle loop) were observed. Collagen, elastic and reticular fibers were there in different components of the kidney. Histochemically it has determined the secretion of neutral as well as acidic mucopolysaccharide substances. PAS activity was observed at mesangial cells and basement membrane of DCT. The Alkaline phosphatase showed the positive activity in capsule, brush border of proximal and distal convoluted tubules. The Acid phosphatases were showing intense activity in parietal and visceral layer of renal corpuscles, lumen and basement membrane of PCT. The intense activity of Succinic dehydrogenase showed at PCT, DCT, collecting tubule whereas renal corpuscles showed feeble activity.

Histomorphological and Histochemical Studies on Proventriculus in Guinea Fowl (Numida meleagris)

Background: The proventriculus, component of digestive system of bird secretes hydrochloric acid and pepsinogen which helps the churning of food material that takes place in ventriculus. Pepsinogen produces pepsin and it smashes the peptide bonds of amino acid of food material. Methods: The twelve proventriculus samples of Guinea fowl birds were fixed in 10% neutral buffered formalin and Bouin’s fluid for histological procedure while chilled acetone and chilled ethanol were used as the fixatives for histochemical studies. The fresh unfixed samples were used on cryostat for studies of succinic dehydrogenase enzyme.Result: Histomorphologically the proventriculus of Guinea fowl was observed as the typical tubular component with four tunics from inside to outside as tunica mucosa, tunica sub mucosa, tunica muscularis and tunica serosa. The submucosal layer comprised with proventricular submucosal gland (PVSMG) which was encapsulated by elastic fibers. Tunica muscularis was inner longitudinal and outer circular layer, while the tunica serosa was observed with surrounding adipose tissue as the connective tissue constituent to endow the access of nutritional supply in the form of blood vessels. The luminal surface of proventricular submucosal glands has shown the significant activity of glycogen while intercalated, intralobular and interlobular ducts have exhibited the weak activity towards the same. The glandular cell rod of PVSMG, serosal layer and adjoining connective tissue with blood vessels has exhibited very weak activity of alkaline phosphatase. Mucosal folds and lumen of PVSMG has shown the weak activity of acidic phosphatase but the serosal limiting membrane has shown very weak activity. The succinic dehydrogenase activity was seen in the connective tissue septa of submucosal glandular cellular rods and the intense activity was observed at tunica muscularis.

The Involvement of Energy Metabolism and Lipid Peroxidation in Lignin Accumulation of Postharvest Pumelos

Lignification is especially prominent in postharvest pumelo fruit, which greatly impairs their attractiveness and commercial value. This study investigated the energy metabolism and lipid peroxidation and their relationship with accumulated lignin content in juice sacs of “Hongroumiyou” (HR) during 90 d of storage at 25 °C. The results indicated that, the alterations of energy metabolism in juice of sacs of postharvest pumelos was featured by a continuous decline in energy charge and ATP/ADP; an increase in succinic dehydrogenase (SDH) activity before 30 d and increases in activities of cytochrome c oxidase (CCO) and F0F1-ATPase before 60 d; but declines in activities of Ca2+-ATPase and H+-ATPase. Additionally, enhanced contents of H2O2, O2−, and –OH scavenging rate; increased malondialdehyde (MDA) content; and transformation of unsaturated fatty acids (USFA) to saturated fatty acids (USFA) and reduced USFA/SFA (U/S) could result in lipid peroxidation and membrane integrity loss. Moreover, correlation analysis showed that lignin accumulation was in close relation to energy metabolism and lipid peroxidation in juice sacs of postharvest pumelos. These results gave evident credence for the involvement of energy metabolism and lipid peroxidation in the lignin accumulation of HR pumelo fruit during postharvest storage.

Dietary Linseed Supplementation Improves Meat Quality of Sheep by Altering Muscle Fiber Characteristics and Antioxidative Capacity

Background: In ruminants, due to the hydrogenation of the rumen, muscles contain a large amount of saturated fatty acids (SFA), which have a negative effect on meat quality. Linseed as a common oil crop which is rich in polyunsaturated fatty acid (PUFA), it affected the deposition of PUFA. Unfortunately, PUFA can exert a negative influence on the oxidative stability of meat. Fortunately, linseed is not only rich in PUFA, but also rich in phenols, which are a good source of antioxidants. Therefore, linseed may be can serve as an effective feed additive to improve meat quality of sheep. The aim of this investigation was to establish the effects of dietary linseed supplementation on carcass traits, meat quality, muscle fiber characteristics and antioxidative capacity of sheep. Results: Results of this study indicated that linseed supplementation significantly increased the loin-eye area, crude protein and ash. Reduced pH24h, L* and shear force. Moreover, linseed supplementation affected the relative content and enriched the kinds of volatile flavor substances. Increased mRNA expression of MyHC I and MyHC IIx, and a decrease in cross-sectional area (CSA) and muscle fiber diameter was also observed. Additional changes included enhanced activity of succinic dehydrogenase (SDH), decreased activity of lactate dehydrogenase (LDH), increased total antioxidative capacity (T-AOC) activity. The mRNA expression of glutathione peroxidase (GSH-PX) and catalase (CAT) were increased while malondialdehyde (MDA) decreased. Conclusions: The results suggest that linseed is an effective feed additive in improving meat quality. The underlying mechanism(s) for its effectiveness may be partly due to a change in muscle fiber characteristics and antioxidative capacity.

THE EFFECT OF MICROWAVES OF A DECIMETER RANGE ON THE FUNCTIONAL ACTIVITY OF MITOCHONDRIA IN DESTRUCTIVE APPENDICITIS IN CHILDREN

Introduction. The article assessed the effectiveness of decimeter wave therapy (DWT) in the postoperative period after laparoscopic appendectomy for destructive appendicitis in children. Authors relate positive clinical effects with the activation of mitochondrial energy metabolism. Material and methods. The study included 132 patients (46 destructive appendicitis cases, 86 h appendicular peritonitis patients). Among them, there were 75 (56.8 percent) boys and 57 (43.2 percent) girls aged of from 3 to 17 years (mean age of 10.7 ± 3.07 years). Patients of the main group received DWT procedures with the use of the apparatus DMV-02 “Solnyshko” starting from the 1st day after the surgery. Patients from the reference group received no physiotherapy treatment. The activity of dehydrogenases of lymphocytes (succinic dehydrogenase (SDH) and NADH-dehydrogenase) in patients from the main group and the comparison group were determined by means of cytomorphological method with the use a hardware-software visualization complex “Videotest” and the “Morphology 5.2” (Russia), at the 1st, 3rd and 5th day of the postoperative period. Results. Under the influence of DWT in the postoperative period the frequency of intestinal insufficiency syndrome, systemic inflammatory response syndrome, infiltrative adhesions was established to decline. There was noted a gain in the activity of mitochondrial enzymes, reflecting the first and second stages of the respiratory chain (SDH and NADH-dehydrogenase). In children with destructive appendicitis against the background of DWT, the normalization of the activity of LDH in moderate activation of NADH-dehydrogenase, and in appendicular peritonitis cases - the significant elevation in LDH activity of lymphocytes and activation of NADH-dehydrogenase occurs. In patients from the main group, the absolute number of peripheral blood lymphocytes normalized Conclusion. Under the influence of DWT there was noted the activation of enzymes of mitochondria that provides a mild course of the postoperative period.

Obesity-induced discrepancy between contractile and metabolic phenotypes in slow- and fast-twitch skeletal muscles of female obese Zucker rats

A clear picture of skeletal muscle adaptations to obesity and related comorbidities remains elusive. This study describes fiber-type characteristics (size, proportions, and oxidative enzyme activity) in two typical hindlimb muscles with opposite structure and function in an animal model of genetic obesity. Lesser fiber diameter, fiber-type composition, and histochemical succinic dehydrogenase activity (an oxidative marker) of muscle fiber types were assessed in slow (soleus)- and fast (tibialis cranialis)-twitch muscles of obese Zucker rats and compared with age (16 wk)- and sex (females)-matched lean Zucker rats ( n = 16/group). Muscle mass and lesser fiber diameter were lower in both muscle types of obese compared with lean animals even though body weights were increased in the obese cohort. A faster fiber-type phenotype also occurred in slow- and fast-twitch muscles of obese rats compared with lean rats. These adaptations were accompanied by a significant increment in histochemical succinic dehydrogenase activity of slow-twitch fibers in the soleus muscle and fast-twitch fiber types in the tibialis cranialis muscle. Obesity significantly increased plasma levels of proinflammatory cytokines but did not significantly affect protein levels of peroxisome proliferator-activated receptors PPARγ or PGC1α in either muscle. These data demonstrate that, in female Zucker rats, obesity induces a reduction of muscle mass in which skeletal muscles show a diminished fiber size and a faster and more oxidative phenotype. It was noteworthy that this discrepancy in muscle's contractile and metabolic features was of comparable nature and extent in muscles with different fiber-type composition and antagonist functions. NEW & NOTEWORTHY This study demonstrates a discrepancy between morphological (reduced muscle mass), contractile (shift toward a faster phenotype), and metabolic (increased mitochondrial oxidative enzyme activity) characteristics in skeletal muscles of female Zucker fatty rats. It is noteworthy that this inconsistency was comparable (in nature and extent) in muscles with different structure and function.

Amelioration of Energy Metabolism by Melatonin in Skeletal Muscle of Rats With LPS Induced Endotoxemia

In the literature, few studies have investigated the effects of melatonin on energy metabolism in skeletal muscle in endotoxemia. We investigated the effects of melatonin on tissue structure, energy metabolism in skeletal muscle, and antioxidant level of rats with endotoxemia. We divided rats into 4 groups, control, lipopolysaccharide (LPS) (20 mg/kg, i.p., single dose), melatonin (10 mg/kg, i.p., three times), and melatonin + LPS. Melatonin was injected i.p. 30 min before and after the 2nd and 4th hours of LPS injection. Antioxidant status was determined by glutathione (GSH) measurement in the blood. Muscle tissue was stained using modified Gomori trichrome (MGT), succinic dehydrogenase (SDH), and cytochrome oxidase (COX) and histological scored. Also the sections were then stained with hematoxylin and eosin. The stained sections were visualized and photographed. Creatine, creatine phosphate, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) levels were investigated using high performance liquid chromatography (HPLC) in muscle tissue. In the Melatonin + LPS group, blood GSH levels were increased compared with the LPS group (P<0.01). Melatonin reduced myopathic changes in the LPS group according to the histopathologic findings. In addition, ATP values were increased compared with the LPS group (P<0.05). Our findings showed melatonin treatment prevented muscle damage by increasing ATP and GSH levels in rats with LPS induced endotoxemia.

Cellular properties of extensor carpi radialis brevis and trapezius muscles in healthy males and females

In this study, we sought to determine whether differences in cellular properties associated with energy homeostasis could explain the higher incidence of work-related myalgia in trapezius (TRAP) compared with extensor carpi radialis brevis (ECRB). Tissue samples were obtained from the ECRB (n = 19) and TRAP (n = 17) of healthy males and females (age 27.9 ± 2.2 and 28.1 ± 1.5 years, respectively; mean ± SE) and analyzed for properties involved in both ATP supply and utilization. The concentration of ATP and the maximal activities of creatine phosphokinase, phosphorylase, and phosphofructokinase were higher (P < 0.05) in ECRB than TRAP. Succinic dehydrogenase, citrate synthase, and cytochrome c oxidase were not different between muscles. The ECRB also displayed a higher concentration of Na+–K+-ATPase and greater sarcoplasmic reticulum Ca2+ release and uptake. No differences existed between muscles for either monocarboxylate transporters or glucose transporters. It is concluded that the potentials for high-energy phosphate transfer, glycogenolysis, glycolysis, and excitation–contraction coupling are higher in ECRB than TRAP. Histochemical measurements indicated that the muscle differences are, in part, related to differing amounts of type II tissue. Depending on the task demands, the TRAP may experience a greater metabolic and excitation–contraction coupling strain than the ECRB given the differences observed.