Dietary Supplementation with 20-Hydroxyecdysone Ameliorates Hepatic Steatosis and Reduces White Adipose Tissue Mass in Ovariectomized Rats Fed a High-Fat, High-Fructose Diet.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is defined as hepatic steatosis in combination with overweight, diabetes, or other metabolic risk factors. MAFLD affects a significant number of the global population and imposes substantial clinical and economic burdens. With no approved pharmacotherapy, current treatment options are limited to diet and exercise. Therefore, the development of medicines for MAFLD treatment or prevention is necessary. 20-Hydroxyecdysone (20E) is a natural steroid found in edible plants and has been shown to improve metabolism and dyslipidemia. Therefore, it may be useful for MAFLD treatment. Here, we aimed to determine how dietary supplementation with 20E affects fat accumulation and lipogenesis in the liver and adipose tissue of ovariectomized rats fed a high-fat, high-fructose diet (OHFFD). We found that 20E reduced hepatic triglyceride content and visceral fat deposition. 20E increased the phosphorylation of AMP-activated protein kinase and acetyl CoA carboxylase while reducing the expression of fatty acid synthase in the liver and adipose tissue. Additionally, 20E increased hepatic expression of carnitine palmitoyltransferase-1 and reduced adipose expression of sterol regulatory element-binding protein-1. In conclusion, 20E demonstrated beneficial effects in rats with OHFFD-induced MAFLD. These findings suggest that 20E may represent a promising option for MAFLD prevention or treatment.

The proximal humeral locking plate positioning to the pectoralis major tendon in achieving the proper calcar screw location: a cadaveric study.

BACKGROUND: Proximal humeral fracture is the third most common of osteoporotic fracture. Most surgical cases were treated by fixation with anatomical locking plate system. The calcar screw plays a role in medial support and improving varus stability. Proximal humerus fracture in elderly patients are commonly seen with greater tuberosity (GT) fracture. The GT fragment is sometimes difficult to use as an anatomic landmark for proper plate and screw position. Therefore, the insertion of pectoralis major tendon (PMT) may be used as an alternative landmark for appropriate plate and calcar screw position. The purpose of study is going to identify the vertical distance from PMT to a definite point on the position of locking plate. METHODS: 30 cadaveric shoulders at the department of clinical anatomy were performed. Shoulders with osteoarthritic change (n = 5) were excluded. Finally, 25 soft cadaveric shoulders were recruited in this study. The PHILOS™ plate was placed 2 mm posterior to the bicipital groove. A humeral head (HH) was cut in the coronal plane at the level of the anterior border of the PHILOS plate with a saw. A calcar screw was inserted close to the inferior cortex of HH. Distance from the upper border of elongated combi-hole (UB-ECH) to the upper border of pectoralis major tendon (UB-PMT) was measured. The plate was then moved superiorly until the calcar screw was 12 mm superior to the inferior border of HH and the distance was repeatedly measured. RESULTS: The range of distance from UB-PMT to the UB-ECH was from - 4.50 ± 7.95 mm to 6.62 ± 7.53 mm, when calcar screw was close to inferior border of HH and when the calcar screw was 12 mm superior to the inferior border of HH, respectively. The highest probability of calcar screw in proper location was 72% when UB-ECH was 3 mm above UB-PMT. DISCUSSION AND CONCLUSION: The GT fragment is sometimes difficult to use as an anatomic landmark for proper plate and screw position. PMT can be used as an alternative anatomic reference. UB-PMT can serve as a guide for proper calcar screw insertion. UB-ECH should be 3 mm above UB-PMT and three-fourths of cases achieved proper calcar screw location.

Morphological alteration of the pancreatic islet in ovariectomized rats fed a high-fat high-fructose diet.

Diabetes and its complications are major causes of mortality worldwide. Type 2 diabetes coexists with insulin resistance and ß-cell dysfunction, which are aggravated by overconsumption and estrogen-deprived conditions. However, the morphology of pancreatic islets in a combined condition of excessive caloric intake and estrogen deficiency has never been described. Herein, we examined morphological changes in the pancreatic islets of ovariectomized (OVX) rats fed a high-fat high-fructose diet (HFFD) for 12 weeks. The histological changes in the size and number of pancreatic islets were assessed by hematoxylin-eosin and immunohistochemical staining. Enlarged pancreatic islets with fat deposition in OVX rats were accompanied by whole-body insulin resistance and hyperglycemia. The addition of a HFFD to OVX rats (OVX + HFFD) further aggravated insulin resistance, with a substantial increase in the density of enlarged pancreatic islets and fat accumulation. The augmented number of enlarged islets was correlated with elevated plasma glucose and insulin levels. Intriguingly, unlike the HFFD and OVX alone, the OVX + HFFD markedly expanded the area of insulin-producing ß-cells and glucagon-producing α-cells. Importantly, enlarged islets, pancreatic fat deposits, and diabetic states developing in OVX + HFFD conditions were resolved by estrogen replacement. Collectively, the morphological characteristics of pancreatic islets were influenced in an insulin-resistant state caused by estrogen deficiency and HFFD consumption and were distinct from each factor alone. A combination of estrogen deficiency with HFFD consumption worsened the integrity of pancreatic islets, ultimately resulting in disease progression. These findings expand our understanding of the causal relationship between pancreatic morphology and diabetes development and suggest therapeutic strategies.

Characterization and tissue distribution of neuropeptide F in the eyestalk and brain of the male giant freshwater prawn, Macrobrachium rosenbergii.

We previously analyzed the central nervous system (CNS) transcriptome and found three isotypes of long neuropeptide F (MrNPF-I, -II, -III) and four isoforms of short NPF (sMrNPF) in the giant freshwater prawn, Macrobrachium rosenbergii. We now validate the complete sequences of the MrNPF-I and -II precursor proteins, which show high similarity (91-95 %) to NPFs of the penaeus shrimp (PsNPF). MrNPF-I and -II precursors share 71 % amino acid identity, whereas the mature 32-amino-acid MrNPF-I and 69-amino-acid MrNPF-II are identical, except for a 37-amino-acid insert within the middle part of the latter. Both mature MrNPFs are almost identical to PsNPF-I and -II except for four amino acids at the mid-region of the peptides. Reverse transcription plus the polymerase chain reaction revealed that transripts of MrNPF-I and -II were expressed in various parts of CNS including the eyestalk, brain and thoracic and abdominal ganglia, with the highest expression occurring in the brain and thoracic ganglia and with MrNPF-I showing five- to seven-fold higher expression than MrNPF-II. These peptides were also expressed in the midgut hindgut, and hepatopancreas, with MrNPF-I expression in the former two organs being at the same level as that in the brain and thoracic ganglia and about 4-fold higher than NPF-II. The expression of NPFs was also detected in the testes and spermatic duct but appeared much weaker in the latter. Other tissues that also expressed a considerable amount of NPF-I included the hematopoeitic tissue, heart and muscle. By immunohistochemistry, we detected MrNPFs in neurons of clusters 2, 3 and 4 and neuropils ME, MT and SG of the optic ganglia, neurons in cluster 6 and neuropils AMPN, PMPN, PT, PB and CB of the medial protocerebrum, neurons in clusters 9 and 11 and neurophils ON and OGTN of the deutocerebrum and neurons in clusters 14, 15 and 16 and neuropils TN and AnN of the tritocerebrum. Because of their high degree of conservation and strong and wide-spread expression in tissues other than CNS, we believe that, in addition to being a neuromodulator in controlling feeding, MrNPFs also play critical roles in tissue homeostasis. This should be further explored.

Effects of andrographolide on intrahepatic cholestasis induced by alpha-naphthylisothiocyanate in rats.

Cholestasis is a cardinal manifestation of liver diseases but effective therapeutic approaches are limited. Therefore, alternative therapy for treating and preventing cholestatic liver diseases is necessary. Andrographolide, a promising anticancer drug derived from the medicinal plant Andrographis paniculata, has diverse pharmacological properties and multi-spectrum therapeutic applications. However, it is unknown whether andrographolide has a hepatoprotective effect on intrahepatic cholestasis. The aims of this study were to investigate the protective effect and possible mechanisms of andrographolide in a rat model of acute intrahepatic cholestasis induced by alpha-naphthylisothiocyanate (ANIT). Andrographolide was administered intragastrically for four consecutive days, with a single intraperitoneal injection of ANIT on the second day. Liver injury was evaluated biochemically and histologically together with hepatic gene and protein expression analysis. Rats pretreated with andrographolide prior to ANIT injection demonstrated lower levels of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, as well as bilirubin and bile acids as compared to rats treated with ANIT alone. Andrographolide also decreased the incidence and extent of periductular fibrosis and bile duct proliferation. Analysis of protein expression in livers from andrographolide-treated cholestatic rats revealed markedly decreased expression of alpha-smooth muscle actin and nuclear factor kappa-B (NF-κB). In conclusion, andrographolide has a potent protective property against ANIT-induced cholestatic liver injury. The mechanisms that underlie this protective effect are mediated through down-regulation of NF-κB expression and inhibition of hepatic stellate cell activation. These findings suggest that andrographolide could be a promising therapeutic option in prevention and slowing down the progression of cholestatic liver diseases.

Gonadotropin-releasing hormone and adipokinetic hormone/corazonin-related peptide in the female prawn.

Crustacean neuropeptides (NPs) play important roles in the regulation of most physiological activities, including growth, molting and reproduction. In this study, we have performed an in silico analysis of female prawn (Macrobrachium rosenbergii) neural transcriptomes to identify NPs not previously identified. We predict that approximately 1309 proteins are destined for the secretory pathway, many of which are likely post-translationally processed to generate active peptides. Within this neural secretome, we identified a gene transcript that encoded a precursor protein with striking similarity to a gonadotropin-releasing hormone (GnRH). We additionally identified another GnRH NP superfamily member, the adipokinetic hormone/corazonin-related peptide (ACP). M. rosenbergii GnRH and ACP were widespread throughout the nervous tissues, implicating them as potential neuromodulators. Furthermore, GnRH was found in non-neural tissues, including the stomach, gut, heart, testis and ovary, in the latter most prominently within secondary oocytes. The GnRH/corazonin receptor-like gene is specific to the ovary, whereas the receptor-like gene expression is more widespread. Administration of GnRH had no effect on ovarian development and maturation, nor any effect on total hemolymph lipid levels, while ACP administration decreased oocyte proliferation (at high dose) and stimulated a significant increase in total hemolymph lipids. In conclusion, our targeted analysis of the M. rosenbergii neural secretome has revealed the decapod GnRH and ACP genes. We propose that ACP in crustaceans plays a role in the lipid metabolism and the inhibition of oocyte proliferation, while the role of the GnRH remains to be clearly defined, possibly through experiments involving gene silencing.

DMPA Suppresses Cell Proliferation and Enhances Cell Apoptosis of Eutopic Endometrium in Women with Endometriosis: A Randomized Controlled Study.

Background: Although Depo-medroxyprogesterone acetate (DMPA), an injectable contraceptive progestin, is very effective for pain relief and prevention of recurrence in women with endometriosis, there is no report on the mechanism of this medication about cell proliferation and apoptosis. Objective: To investigate the effects of DMPA on cell proliferation and apoptosis in the eutopic endometrium of women with endometriosis. Material and Method: A randomized controlled study was conducted in 28 women with endometriosis. The DMPA-treated group included 14 women who were scheduled to undergo laparoscopic surgery after 150 mg of DMPA injections. The control group included 14 women who were scheduled to undergo the surgery without DMPA injection. The endometrial tissue was obtained from each woman by endometrial aspiration before surgery. The ELISA formats of PCNA and the quantitative colorimetric analysis of TUNEL were used for estimating cell proliferation and apoptosis of the eutopic endometrium. Results: There were no differences in the women characteristics between the two groups. The relative level of cell proliferation was significantly less in the DMPA than the control groups (1.08±0.57 vs. 1.73±0.50, p = 0.014). Whereas the relative level of cell apoptosis was greater in the DMPA group than that in the control group (1.12±0.36 vs. 0.82±0.39, p = 0.034). Conclusion: Three months of 150 mg DMPA treatment could suppress cell proliferation and enhance cell apoptosis of the eutopic endometrium of women with endometriosis.

Nuclear Factor, Erythroid 2-Like 2 Regulates Expression of Type 3 Inositol 1,4,5-Trisphosphate Receptor and Calcium Signaling in Cholangiocytes.

BACKGROUND & AIMS: Most cholestatic disorders are caused by defects in cholangiocytes. The type 3 isoform of the inositol 1,4,5-trisphosphate receptor (ITPR3) is the most abundant intracellular calcium release channel in cholangiocytes. ITPR3 is required for bicarbonate secretion by bile ducts, and its expression is reduced in intrahepatic bile ducts of patients with cholestatic disorders. We investigated whether the nuclear factor, erythroid 2-like 2 (NFE2L2 or NRF2), which is sensitive to oxidative stress, regulates expression of ITPR3. METHODS: The activity of the ITPR3 promoter was measured in normal human cholangiocyte (NHC) cells and primary mouse cholangiocytes. Levels of ITPR3 protein and messenger RNA were examined by immunoblot and polymerase chain reaction analyses, respectively. ITPR3 activity was determined by measuring calcium signaling in normal human cholangiocyte cells and secretion in isolated bile duct units. Levels of NRF2 were measured in liver tissues from rats with cholestasis (induced by administration of α-napthylisothiocyanate) and from patients with biliary diseases. RESULTS: We identified a musculo-aponeurotic fibrosarcoma recognition element in the promoter of ITPR3 that bound NRF2 directly in NHC cells and mouse cholangiocytes. Increasing binding of NRF2 at this site resulted in chromatin remodeling that reduced promoter activity. Mutant forms of the musculo-aponeurotic fibrosarcoma recognition element did not bind NRF2. Activation of NRF2 with quercetin or by oxidative stress reduced expression of ITPR3 and calcium signaling in NHC cells; quercetin also reduced secretion by bile duct units isolated from rats. Knockdown of NRF2 with small interfering RNAs restored expression and function of ITPR3 in NHC cells incubated with quercetin. Bile ducts from rats with cholestasis and patients with cholangiopathic disorders expressed higher levels of NRF2 and lower levels of ITPR3 than ducts from control rats or patients with other liver disorders. CONCLUSIONS: The transcription factor NRF2 binds to the promoter of ITPR3 to inhibit its expression in cholangiocytes, leading to reduced calcium signaling and bile duct secretion. This could be a mechanism by which oxidative stress inhibits these processes and contributes to cholangiopathies.

Changes of phosphatidylcholine and fatty acids in germ cells during testicular maturation in three developmental male morphotypes of Macrobrachium rosenbergii revealed by imaging mass spectrometry.

Testis maturation, germ cell development and function of sperm, are related to lipid composition. Phosphatidylcholines (PCs) play a key role in the structure and function of testes. As well, increases of polyunsaturated fatty acids (PUFA) and highly unsaturated fatty acids (HUFA), especially arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are essential for male fertility. This study is the first report to show the composition and distribution of PCs and total fatty acids (FAs) in three groups of seminiferous tubules (STs) classified by cellular associations [i.e., A (STs with mostly early germ cells), B (STs with mostly spermatids), and C (STs with spermatozoa)], in three morphotypes of Macrobrachium rosenbergii, [i.e., small male (SM), orange claw male (OC), and blue claw male (BC)]. Thin layer chromatography exhibited levels of PCs reaching maxima in STs of group B. Imaging mass spectrometry showed remarkably high signals corresponding to PC (16:0/18:1), PC (18:0/18:2), PC (18:2/20:5), and PC (16:0/22:6) in STs of groups A and B. Moreover, most signals were detected in the early developing cells and the intertubular area, but not at the area containing spermatozoa. Finally, gas chromatography-mass spectrometry indicated that the major FAs present in the testes were composed of 14:0, 16:0, 17:0, 18:0, 16:1, 18:1, 18:2, 20:1, 20:2, 20:4, 20:5, and 22:6. The testes of OC contained the greatest amounts of these FAs while the testes of BC contained the least amounts of these FAs, and there was more EPA (20:5) in the testes of SM and OC than those in the BC. The increasing amounts of FAs in the SM and OC indicate that they are important for spermatogenesis and spermiogenesis. This knowledge will be useful in formulating diets containing PUFA and HUFA for prawn broodstocks in order to improve testis development, and lead to increased male fecundity.

The in vitro anthelmintic effects of plumbagin on newly excysted and 4-weeks-old juvenile parasites of Fasciola gigantica.

The effect of plumbagin (PB, 5-hydroxy-2-methyl-1,4-naphthoquinone) against newly excysted juveniles (NEJs) and 4-weeks-old immature parasites of Fasciola gigantica were compared with triclabendazole (TCZ). The anthelmintic efficacy of 1, 10 and 100µg/ml of PB or TCZ following incubation in vitro for 1-24h was compared using a combination of relative motility (RM), survival index (SI) and larval migration inhibition (LMI) assays for parasite viability. The RM and SI values of the PB-treated group decreased at a more rapid rate than the TCZ-treated group. For NEJs, the decreased RM values were first observed at 1h incubation with 1µg/ml PB, and 90% of flukes were killed at 24h. In contrast, in TCZ-treated groups a 10-fold higher concentration of TCZ (10µg/ml) resulted in only 9% dead parasites after 24h incubation. In 4-weeks-old juvenile parasites, PB reduced the RM value at 10µg/ml with 100% of flukes dead after 3h, while TCZ decreased RM values at the concentration of 100µg/ml but with only 5% of flukes killed at 24h. NEJs treated with PB exhibited 88%, 99% and 100% of LMIs at the concentrations of 1, 10 and 100µg/ml, respectively. NEJs incubated with TCZ have an LMI of only 32% at the highest concentration of 100µg/ml. Similarly PB had a significantly greater killing of immature 4weeks juvenile stages than TCZ at all concentrations; however, 4-weeks-old juvenile parasites were more resistant to killing by PB or TCZ at all concentrations when compared to NEJs. Further studies were carried out to investigate the alterations of the parasite tegument by scanning electron microscope (SEM). PB caused similar tegumental alterations in 4-weeks-old juveniles as those observed in TCZ treatment but with greater damage at comparative time points, comprising of swelling, blebbing and rupture of the tegument, loss of spines, and eventual erosion, lesion and desquamation of the total tegument. These data indicate that PB had a greater fasciolicidal effect against immature stages of F. gigantica parasites than TCZ and warrant further studies for use as a potential new anthelmintic against Fasciola infections.

Characterization of red pigment concentrating hormone (RPCH) in the female mud crab (Scylla olivacea) and the effect of 5-HT on its expression.

Red pigment concentrating hormone (RPCH) is a member of the chromatophorotropic hormones and, in crustaceans, it is synthesized in the eyestalk. We have isolated a full-length cDNA for a RPCH preprohormone gene (Scyol-RPCH) from the eyestalks of female mud crabs, Scylla olivacea. The open reading frame consists of 642 nucleotides, and encodes a deduced 108 amino acid precursor protein, which includes a signal peptide, the RPCH (pQLNFSPGWamide), and an associated peptide. We show that the mud crab RPCH peptide exhibits 100% identity with 15 other decapods. Expression of Scyol-RPCH within adult mud crab takes place in the eyestalk, brain, and ventral nerve cord, comprising subesophageal ganglion, thoracic ganglion, and abdominal ganglion. In situ hybridization demonstrates specific expression within neuronal clusters 1, 2, 3, and 4 of the eyestalk X-organ, clusters 6, 8, 9, 10, and 17 of the brain, and in neuronal clusters of the ventral nerve cord. We found that administration of 5-HT up-regulates RPCH gene expression in the eyestalk, suggesting that RPCH may play a role as a downstream hormone of 5-HT.

Composition and localization of lipids in Penaeus merguiensis ovaries during the ovarian maturation cycle as revealed by imaging mass spectrometry.

Ovary maturation, oocyte differentiation, and embryonic development in shrimp are highly dependent on nutritional lipids taken up by female broodstocks. These lipids are important as energy sources as well as for cell signaling. In this study, we report on the compositions of major lipids, i.e. phosphatidylcholines (PCs), triacylglycerols (TAGs), and fatty acids (FAs), in the ovaries of the banana shrimp, Penaeus merguiensis, during ovarian maturation. Thin-layer chromatography analysis showed that the total PC and TAG signal intensities increased during ovarian maturation. Further, by using gas chromatography, we found that (1) FAs 14:0, 16:1, 18:1, 18:2, 20:1, and 22:6 proportionally increased as ovarian development progressed to more mature stages; (2) FAs 16:0, 18:0, 20:4, and 20:5 proportionally decreased; and (3) FAs 15:0, 17:0, and 20:2 remained unchanged. By using imaging mass spectrometry, we found that PC 16:0/16:1 and TAG 18:1/18:2/22:6 were detected in oocytes stages 1 and 2. PCs 16:1/20:4, 16:0/22:6, 18:3/22:6, 18:1/22:6, 20:5/22:6, and 22:6/22:6 and TAGs 16:0/16:1/18:3, 16:0/18:1/18:3, 16:0/18:1/18:1, and 16:0/18:2/22:6 were present in all stages of oocytes. In contrast, the PC- and TAG-associated FAs 20:4, 20:5, and 22:6 showed high signal intensities in stage 3 and 4 oocytes. These FAs may act as nutrition sources as well as signaling molecules for developing embryos and the hatching process. Knowledge of lipid compositions and localization could be helpful for formulating the diet for female broodstocks to promote fecundity and larval production.

Visualization of neuropeptides in paraffin-embedded tissue sections of the central nervous system in the decapod crustacean, Penaeus monodon, by imaging mass spectrometry.

The distributions of neuropeptides in paraffin-embedded tissue sections (PETS) of the eyestalk, brain, and thoracic ganglia of the shrimp Penaeus monodon were visualized by imaging mass spectrometry (IMS). Peptide signals were obtained from PETS without affecting morphological features. Twenty-nine neuropeptides comprising members of FMRFamide, SIFamides, crustacean hyperglycaemic hormone, orcokinin-related peptides, tachykinin-related peptides, and allatostatin A were detected and visualized. Among these findings we first identified tachykinin-related peptide as a novel neuropeptide in this shrimp species. We found that these neuropeptides were distributed at specific areas in the three neural organs. In addition, 28 peptide sequences derived from 4 types of constitutive proteins, including actin, histones, arginine kinase, and cyclophilin A were also detected. All peptide sequences were verified by liquid chromatography-tandem mass spectrometry. The use of IMS on acetic acid-treated PETS enabled us to identify peptides and obtain their specific localizations in correlation with the undisturbed histological structure of the tissue samples.