Long-Term Supplementation of Royal Jelly (Raydel®) Improves Zebrafish Growth, Embryo Production and Survivability, Blood Lipid Profile and Functionality of Vital Organs: A 72-Weeks' Consumption Study.

Royal jelly is a honeybee product with substantial pharmacological and health promotional activities. Nevertheless, the health implications associated with the prolonged dietary supplementation of royal jelly have yet to be elucidated extensively. Herein, 72 weeks of dietary supplementation of royal jelly at 5% and 10% (w/w) were investigated to assess the impact on zebrafish survivability, body weight, liver, testis, ovary functionality, and blood lipid profile. The results revealed no adverse effect of 72 weeks of royal jelly supplementation on zebrafish survivability. Conversely, a noteworthy enhancement in the zebrafish body weight was observed in royal-jelly-supplemented zebrafish in a concentration-dependent manner [5% and 10% (w/w)]. Interestingly, female zebrafish were found to be more biased, with a significant 17% (p < 0.001) and 23% (p < 0.001) higher body weight enhancement after 72 weeks of consumption of 5% and 10% (w/w) royal jelly, compared to the male zebrafish. The histological outcome revealed no sign of hepatotoxicity; moreover, diminished reactive oxygen species (ROS) and apoptosis were observed in the hepatic tissue of the royal-jelly-supplemented group. Consistent with the histological outcomes, the liver function biomarkers, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), exhibited a significant decrease of 1.9-fold (p = 0.006) and 1.4-fold (p = 0.003) in zebrafish supplemented with royal jelly compared to those on a normal diet (ND) and zebrafish given supplements. Also, no sign of ovary and testis-related toxicity was observed in the royal-jelly-supplemented group during the 72-week period. Furthermore, the 10% (w/w) royal-jelly-consuming zebrafish exhibited a notable 2.1-fold increase (p = 0.018) in egg-laying ability compared to the ND-supplemented zebrafish. The 10% (w/w) royal jelly supplementation also effectively maintained the blood lipid profile by curtailing serum triglycerides (TG) and elevating high-density lipoprotein cholesterol (HDL-C). Conclusively, royal jelly dietary supplementation for a prolonged time found royal jelly to be safe to consume, to efficiently improve hepatic function, reproduction, and sexual health, and to augment the serum HDL-C level.

Beeswax Alcohol Prevents Low-Density Lipoprotein Oxidation and Demonstrates Antioxidant Activities in Zebrafish Embryos and Human Subjects: A Clinical Study.

Oxidative stress is one of the primary instigators of the onset of various human ailments, including cancers, cardiovascular diseases, and dementia. Particularly, oxidative stress severely affects low-density lipid & protein (LDL) oxidation, leading to several detrimental health effects. Therefore, in this study, the effect of beeswax alcohol (BWA) was evaluated in the prevention of LDL oxidation, enhancement of paraoxonase 1 (PON-1) activity of high-density lipid & protein (HDL), and zebrafish embryo survivability. Furthermore, the implication of BWA consumption on the oxidative plasma variables was assessed by a preliminary clinical study on middle-aged and older human subjects (n = 50). Results support BWA augmentation of PON-1 activity in a dose-dependent manner (10-30 µM), which was significantly better than the effect exerted by coenzyme Q10 (CoQ10). Moreover, BWA significantly curtails LDL/apo-B oxidation evoked by CuSO4 (final 0.5 µM) and a causes a marked reduction in lipid peroxidation in LDL. The transmission electron microscopy (TEM) analysis revealed a healing effect of BWA towards the restoration of LDL morphology and size impaired by the exposure of Cu2+ ions (final 0.5 µM). Additionally, BWA counters the toxicity induced by carboxymethyllysine (CML, 500 ng) and rescues zebrafish embryos from development deformities and apoptotic cell death. A completely randomized, double-blinded, placebo-controlled preliminary clinical study on middle- and older-aged human subjects (n = 50) showed that 12 weeks of BWA (100 mg/day) supplementation efficiently diminished serum malondialdehyde (MDA) and total hydroperoxides and enhanced total antioxidant status by 25%, 27%, and 22%, respectively, compared to the placebo-control and baseline values. Furthermore, the consumption of BWA did not exhibit any noteworthy changes in physical variables, lipid profile, glucose levels, and biomarkers pertinent to kidney and liver function, thus confirming the safety of BWA for consumption. Conclusively, in vitro, BWA prevents LDL oxidation, enhances PON-1 activity in HDL, and positively influences oxidative variables in human subjects.

Combination Therapy of Cuban Policosanol (Raydel®, 20 mg) and Intensive Exercise for 12 Weeks Resulted in Improvements in Obesity, Hypertension, and Dyslipidemia without a Decrease in Serum Coenzyme Q10: Enhancement of Lipoproteins Quality and Antioxidant Functionality in Obese Participants.

Obesity and overweight, frequently caused by a lack of exercise, are associated with many metabolic diseases, such as hypertension, diabetes, and dyslipidemia. Aerobic exercise effectively increases the high-density lipoproteins-cholesterol (HDL-C) levels and alleviates the triglyceride (TG) levels. The consumption of Cuban policosanol (Raydel®) is also effective in enhancing the HDL-C quantity and HDL functionality to treat dyslipidemia and hypertension. On the other hand, no study has examined the effects of a combination of high-intensity exercise and policosanol consumption in obese subjects to improve metabolic disorders. In the current study, 17 obese subjects (average BMI 30.1 ± 1.1 kg/m2, eight male and nine female) were recruited to participate in a program combining exercise and policosanol (20 mg) consumption for 12 weeks. After completion, their BMI, waist circumference, total fat mass, systolic blood pressure (SBP), and diastolic blood pressure (DBP) reduced significantly up to around -15%, -13%, -33%, -11%, and -13%, respectively. In the serum lipid profile, at Week 12, a significant reduction was observed in the total cholesterol (TC) and triglyceride (TG) levels, up to -17% and -54% from the baseline, respectively. The serum HDL-C was elevated by approximately +12% from the baseline, as well as the percentage of HDL-C in TC, and HDL-C/TC (%), was enhanced by up to +32% at Week 12. The serum coenzyme Q10 (CoQ10) level was increased 1.2-fold from the baseline in all participants at Week 12. In particular, the male participants exhibited a 1.4-fold increase from the baseline. The larger rise in serum CoQ10 was correlated with the larger increase in the serum HDL-C (r = 0.621, p = 0.018). The hepatic function parameters were improved; the serum γ-glutamyl transferase decreased at Week 12 by up to -55% (p < 0.007), while the aspartate aminotransferase and alanine transaminase levels diminished within the normal range. In the lipoprotein level, the extent of oxidation and glycation were reduced significantly with the reduction in TG content. The antioxidant abilities of HDL, such as paraoxonase (PON) and ferric ion reduction ability (FRA), were enhanced significantly by up to 1.8-fold and 1.6-fold at Week 12. The particle size and number of HDL were elevated up to +10% during the 12 weeks, with a remarkable decline in the TG content, glycation extent, and oxidation. The improvements in HDL quality and functionality were linked to the higher survivability of adult zebrafish and their embryos, under the co-presence of carboxymethyllysine (CML), a pro-inflammatory molecule known to cause acute death. In conclusion, 12 weeks of Cuban policosanol (Raydel®, 20 mg) consumption with high-intensity exercise displayed a significant improvement in blood pressure, body fat mass, blood lipid profile without liver damage, CoQ10 metabolism, and renal impairment.

Enhancement of Antioxidant and Anti-Glycation Properties of Beeswax Alcohol in Reconstituted High-Density Lipoprotein: Safeguarding against Carboxymethyllysine Toxicity in Zebrafish.

The antioxidant and anti-inflammatory abilities of beeswax alcohol (BWA) are well reported in animal and human clinical studies, with a significant decrease in malondialdehyde (MDA) in the blood, reduced liver steatosis, and decreased insulin. However, there has been insufficient information to explain BWAs in vitro antioxidant and anti-inflammatory activity owing to its limited solubility in an aqueous buffer system. Herein, three distinct reconstituted high-density lipoproteins (rHDL) were prepared with palmitoyloleoyl phosphatidylcholine (POPC), cholesterol, apolipoprotein A-I (apoA-I), and BWA at molar ratios of 95:5:1:0 (rHDL-0), 95:5:1:0.5 (rHDL-0.5), and 95:5:1:1 (rHDL-1) and examined for antioxidant and anti-glycation effects. A rHDL containing BWA, precisely rHDL-1, displayed a remarkable anti-glycation effect against fructose (final 250 mM), induced glycation of HDL, and prevented proteolytic degradation of apoA-I. Also, BWA incorporated rHDL-0.5, and rHDL-1 displayed substantial antioxidant activity by inhibiting cupric ion-mediated low-density lipoprotein (LDL) oxidation. In contrast to rHDL-0, a 20 and 22% enhancement in ferric ion reduction ability (FRA) and paraoxonase (PON) activity was observed in HDL treated with rHDL-1, signifying the effect of BWA on the antioxidant activity enhancement of HDL. rHDL-1 efficiently inhibits Nε-carboxylmethyllysine (CML)-induced reactive oxygen species (ROS) generation and apoptosis in zebrafish embryos, consequently improving embryo survivability and developmental deformities impaired by the CML. The dermal application of rHDL-1 to the CML-impaired cutaneous wound of the adult zebrafish inhibited ROS production and displayed potent wound-healing activity. Conclusively, incorporating BWA in rHDL significantly enhanced the anti-glycation and antioxidant activities in rHDL via more stabilization of apoA-I with a larger particle size. The rHDL containing BWA facilitated the inherent antioxidant ability of HDL to suppress the CML-induced toxicities in zebrafish embryos and ameliorate CML-aggravated chronic wounds in adult zebrafish.

Comparison of Policosanols via Incorporation into Reconstituted High-Density Lipoproteins: Cuban Policosanol (Raydel®) Exerts the Highest Antioxidant, Anti-Glycation, and Anti-Inflammatory Activity.

Reconstituted high-density lipoproteins (rHDL) containing each policosanol from Cuba (Raydel®), China (Shaanxi Pioneer), and the United States (Lesstanol®) were synthesized to compare the physiological properties of policosanol depending on sources and origin countries. After synthesis with apolipoproteinA-I (apoA-I) into rHDL, all policosanols bound well with phospholipid and apoA-I to form discoidal rHDL. An rHDL containing Cuban policosanol (rHDL-1) showed the largest rHDL particle size of around 83 ± 3 nm, while rHDL containing Chinese policosanol (rHDL-2) or American policosanol (rHDL-3) showed smaller particles around 63 ± 3 nm and 60 ± 2 nm in diameter, respectively. The rHDL-1 showed the strongest anti-glycation activity to protect the apoA-I degradation of HDL from fructose-mediated glycation: approximately 2.7-times higher ability to suppress glycation and 1.4-times higher protection ability of apoA-I than that of rHDL-2 and rHDL-3. The rHDL-1 showed the highest antioxidant ability to inhibit cupric ion-mediated LDL oxidation in electromobility and the quantification of oxidized species. A microinjection of each rHDL into a zebrafish embryo in the presence of carboxymethyllysine (CML) showed that rHDL-1 displayed the strongest anti-oxidant activity with the highest embryo survivability, whereas rHDL-2 and rHDL-3 showed much weaker protection ability, similar to rHDL alone (rHDL-0). An intraperitoneal injection of CML (250 µg) into adult zebrafish caused acute death and hyperinflammation with an elevation of infiltration of neutrophils and IL-6 production in the liver. On the other hand, a co-injection of rHDL-1 resulted in the highest survivability and the strongest anti-inflammatory ability to suppress IL-6 production with an improvement of the blood lipid profile, such as elevation of HDL-C and lowering of the total cholesterol, LDL-cholesterol, and triglyceride. In conclusion, Cuban policosanol exhibited the most desirable properties for the in vitro synthesis of rHDL with the stabilization of apoA-I, the largest particle size, anti-glycation against fructation, and antioxidant activities to prevent LDL oxidation. Cuban policosanol in rHDL also exhibited the strongest in vivo antioxidant and anti-inflammatory activities with the highest survivability in zebrafish embryos and adults via the prevention of hyperinflammation in the presence of CML.

CIGB-258 Exerts Potent Anti-Inflammatory Activity against Carboxymethyllysine-Induced Acute Inflammation in Hyperlipidemic Zebrafish via the Protection of Apolipoprotein A-I.

Inflammation and atherosclerosis are intimately associated via the production of dysfunctional high-density lipoproteins (HDL) and modification of apolipoprotein (apo) A-I. A putative interaction between CIGB-258 and apoA-I was investigated to provide mechanistic insight into the protection of HDL. The protective activity of CIGB-258 was tested in the CML-mediated glycation of apoA-I. The in vivo anti-inflammatory efficacy was compared in paralyzed hyperlipidemic zebrafish and its embryo in the presence of CML. Treatment of CML induced greater glycation extent of HDL/apoA-I and proteolytic degradation of apoA-I. In the presence of CML, however, co-treatment of CIGB-258 inhibited the glycation of apoA-I and protected the degradation of apoA-I, exerting enhanced ferric ion reduction ability. Microinjection of CML (500 ng) into zebrafish embryos resulted in acute death with the lowest survivability with severe developmental defects with interleukin (IL)-6 production. Conversely, a co-injection of CIGB-258 or Tocilizumab produced the highest survivability with a normal development speed and morphology. In hyperlipidemic zebrafish, intraperitoneal injection of CML (500 µg) caused the complete loss of swimming ability and severe acute death with only 13% survivability 3 h post-injection. A co-injection of the CIGB-258 resulted in a 2.2-fold faster recovery of swimming ability than CML alone, with higher survivability of approximately 57%. These results suggest that CIGB-258 protected hyperlipidemic zebrafish from the acute neurotoxicity of CML. Histological analysis showed that the CIGB-258 group had 37% lower infiltration of neutrophils in hepatic tissue and 70% lower fatty liver changes than those of the CML-alone group. The CIGB-258 group exhibited the smallest IL-6 expression in the liver and the lowest blood triglyceride level. CIGB-258 displayed potent anti-inflammatory activity in hyperlipidemic zebrafish by inhibiting apoA-I glycation, promoting rapid recovery from the paralysis of CML toxicity and suppression of IL-6, and lowering fatty liver changes.

Beneficial Effect of Cuban Policosanol on Blood Pressure and Serum Lipoproteins Accompanied with Lowered Glycated Hemoglobin and Enhanced High-Density Lipoprotein Functionalities in a Randomized, Placebo-Controlled, and Double-Blinded Trial with Healthy Japanese.

This study evaluated the efficacy and safety of 20 mg of Cuban policosanol in blood pressure (BP) and lipid/lipoprotein parameters of healthy Japanese subjects via a placebo-controlled, randomized, and double-blinded human trial. After 12 weeks of consumption, the policosanol group showed significantly lower BP, glycated hemoglobin (HbA1c), and blood urea nitrogen (BUN) levels. The policosanol group also showed lower aspartate aminotransferase (AST), alanine aminotransferase (ALT), and γ-glutamyl transferase (γ-GTP) levels at week 12 than those at week 0: A decrease of up to 9% (p < 0.05), 17% (p < 0.05), and 15% (p < 0.05) was observed, respectively. The policosanol group showed significantly higher HDL-C level and HDL-C/TC (%), approximately 9.5% (p < 0.001) and 7.2% (p = 0.003), respectively, than the placebo group and a difference in the point of time and group interaction (p < 0.001). In lipoprotein analysis, the policosanol group showed a decrease in oxidation and glycation extent in VLDL and LDL with an improvement of particle shape and morphology after 12 weeks. HDL from the policosanol group showed in vitro stronger antioxidant and in vivo anti-inflammatory abilities. In conclusion, 12 weeks of Cuban policosanolconsumption in Japanese subjects showed significant improvement in blood pressure, lipid profiles, hepatic functions, and HbA1c with enhancement of HDL functionalities.

Cuban Sugar Cane Wax Alcohol Exhibited Enhanced Antioxidant, Anti-Glycation and Anti-Inflammatory Activity in Reconstituted High-Density Lipoprotein (rHDL) with Improved Structural and Functional Correlations: Comparison of Various Policosanols.

Policosanols from various sources, such as sugar cane, rice bran, and insects, have been marketed to prevent dyslipidemia, diabetes, and hypertension by increasing the blood high-density lipoproteins cholesterol (HDL-C) levels. On the other hand, there has been no study on how each policosanol influences the quality of HDL particles and their functionality. Reconstituted high-density lipoproteins (rHDLs) with apolipoprotein (apo) A-I and each policosanol were synthesized using the sodium cholate dialysis method to compare the policosanols in lipoprotein metabolism. Each rHDL was compared regarding the particle size and shape, antioxidant activity, and anti-inflammatory activity in vitro and in zebrafish embryos. This study compared four policosanols including one policosanol from Cuba (Raydel® policosanol) and three policosanols from China (Xi'an Natural sugar cane, Xi'an Realin sugar cane, and Shaanxi rice bran). The synthesis of rHDLs with various policosanols (PCO) from Cuba or China using a molar ratio of 95:5:1:1 with palmitoyloleoyl phosphatidylcholine (POPC): free cholesterol (FC): apoA-I:PCO (wt:wt) showed that rHDL containing Cuban policosanol (rHDL-1) showed the largest particle size and the most distinct particle shape. The rHDL-1 showed a 23% larger particle diameter and increased apoA-I molecular weight with a 1.9 nm blue shift of the maximum wavelength fluorescence than rHDL alone (rHDL-0). Other rHDLs containing Chinese policosanols (rHDL-2, rHDL-3, and rHDL-4) showed similar particle sizes with an rHDL-0 and 1.1-1.3 nm blue shift of wavelength maximum fluorescence (WMF). Among all rHDLs, the rHDL-1 showed the strongest antioxidant ability to inhibit cupric ion-mediated LDL oxidation. The rHDL-1-treated LDL showed the most distinct band intensity and particle morphology compared with the other rHDLs. The rHDL-1 also exerted the highest anti-glycation activity to inhibit the fructose-mediated glycation of human HDL2 with the protection of apoA-I from proteolytic degradation. At the same time, other rHDLs showed a loss of anti-glycation activity with severe degradation. A microinjection of each rHDL alone showed that rHDL-1 had the highest survivability of approximately 85 ± 3%, with the fastest developmental speed and morphology. In contrast, rHDL-3 showed the lowest survivability, around 71 ± 5%, with the slowest developmental speed. A microinjection of carboxymethyllysine (CML), a pro-inflammatory advanced glycated end product, into zebrafish embryos resulted in severe embryo death of approximately 30 ± 3% and developmental defects with the slowest developmental speed. On the other hand, the phosphate buffered saline (PBS)-injected embryo showed 83 ± 3% survivability. A co-injection of CML and each rHDL into adult zebrafish showed that rHDL-1 (Cuban policosanol) induced the highest survivability, around 85 ± 3%, while rHDL-0 showed 67 ± 7% survivability. In addition, rHDL-2, rHDL-3, and rHDL-4 showed 67 ± 5%, 62 ± 37, and 71 ± 6% survivability, respectively, with a slower developmental speed and morphology. In conclusion, Cuban policosanol showed the strongest ability to form rHDLs with the most distinct morphology and the largest size. The rHDL-containing Cuban policosanol (rHDL-1) showed the strongest antioxidant ability against LDL oxidation, anti-glycation activity to protect apoA-I from degradation, and the highest anti-inflammatory activity to protect embryo death under the presence of CML.

Enhancement of High-Density Lipoprotein (HDL) Quantity and Quality by Regular and Habitual Exercise in Middle-Aged Women with Improvements in Lipid and Apolipoprotein Profiles: Larger Particle Size and Higher Antioxidant Ability of HDL.

Regular exercise, especially aerobic exercise, is beneficial for increasing serum high-density lipoprotein-cholesterol (HDL-C) levels in the general population. In addition to the HDL-C quantity, exercise enhances HDL functionality, antioxidants, and cholesterol efflux. On the other hand, the optimal intensity and frequency of exercise to increase HDL quantity and enhance HDL quality in middle-aged women need to be determined. The current study was designed to compare the changes in HDL quantity and quality among middle-aged women depending on exercise intensity, frequency, and duration; participants were divided into a sedentary group (group 1), a middle-intensity group (group 2), and a high-intensity group (group 3). There were no differences in anthropometric parameters among the groups, including blood pressure, muscle mass, and handgrip strength. Although there was no difference in serum total cholesterol (TC) among the groups, the serum HDL-C and apolipoprotein (apo)A-I levels remarkably increased to 17% and 12%, respectively, in group 3. Serum low-density lipoprotein-cholesterol (LDL-C), glucose, triglyceride, and the apo-B/apoA-I ratio were remarkably decreased in the exercise groups depending on the exercise intensity; group 3 showed 13%, 10%, and 45% lower LDL-C, glucose, and triglyceride (TG), respectively, than group 1. The hepatic and muscle damage parameter, aspartate aminotransferase (AST), was significantly decreased in the exercise groups, but high-sensitivity C-reactive protein (CRP), alanine aminotransferase (ALT), and γ-glutamyl transferase (γ-GTP) were similar in the three groups. In LDL, the particle size was increased 1.5-fold (p < 0.001), and the oxidation extent was decreased by 40% with a 23% lower TG content in group 3 than in group 1. In the exercise groups (groups 2 and 3), LDL showed the slowest electromobility with a distinct band intensity compared to the sedentary group (group 1). In HDL2, the particle size was 2.1-fold increased (p < 0.001) in the exercise group (group 3) with a 1.5-fold increase in TC content compared to that in group 1, as well as significantly enhanced antioxidant abilities, paraoxonase (PON) activity, and ferric ion reduction ability (FRA). In HDL3, the particle size was increased 1.2-fold with a 45% reduction in TG in group 3 compared to group 1. With increasing exercise intensity, apoA-I expression was increased in HDL2 and HDL3, and PON activity and FRA were enhanced (p < 0.001). In conclusion, regular exercise in middle-aged women is associated with the elevation of serum HDL-C and apoA-I with the enhancement of HDL quality and functionality and an increase in the TC content, particle size, and antioxidant abilities. With the reduction in TG and oxidized products in LDL and HDL, lipoproteins could have more anti-atherogenic properties through regular exercise in an intensity-dependent manner.

Consumption of Policosanol (Raydel®) Improves Hepatic, Renal, and Reproductive Functions in Zebrafish: In Vivo Comparison Study among Cuban, Chinese, and American Policosanol.

The current study compared three policosanols from Cuba (sugarcane, Raydel®, policosanol (1), China (rice bran, Shaanxi, policosanol (2), and the USA (sugarcane, Lesstanol®, policosanol (3) in the treatment of dyslipidemia and protection of the liver, ovary, and testis in hypercholesterolemic zebrafish. After twelve weeks of supplementation of each policosanol (PCO, final 0.1% in diet, w/w) with a high cholesterol diet (HCD, final 4%, w/w), the Raydel policosanol (PCO1) group showed the highest survivability, approximately 89%. In contrast, Shaanxi policosanol (PCO2) and Lesstanol policosanol (PCO3) produced 73% and 87% survivability, respectively, while the HCD alone group showed 75% survivability. In the 12th week, the PCO1 group demonstrated the most modest increase in body weight along with significantly lower levels of total cholesterol (TC) and triglycerides (TG) in comparison to the HCD control group. Additionally, the PCO1 group exhibited the highest proportion of high-density lipoprotein (HDL)-cholesterol within TC. Notably, the PCO1 group displayed the lowest level of aspartate aminotransferase and alanine aminotransferase, minimal infiltration of inflammatory cells, reduced interleukin (IL)-6 production in the liver, a notable decline in reactive oxygen species (ROS) generation and mitigated fatty liver changes. HCD supplementation induced impairment of kidney morphology with the greatest extent of ROS production and apoptosis. On the other hand, the PCO 1 group showed a remarkably improved morphology with the least ROS generation and apoptosis. Within the ovarian context, the PCO1 group exhibited the most substantial presence of mature vitellogenic oocytes, accompanied by minimal levels of ROS and apoptosis. Similarly, in the testicular domain, the PCO1 group showcased optimal morphology for spermatogenesis, characterized by the least interstitial area and diminished production of ROS in testicular cells. At week 8, the PCO1 group showed the highest egg-laying ability, with around 244 eggs produced per mating. In contrast, the HCD alone, PCO2, and PCO3 groups showed significantly lower egg-laying ability (49, 59, and 86 eggs, respectively). The embryos from the PCO1 group exhibited the highest survivability with the fastest swimming ability and developmental speed. These results suggest that PCO1 consumption significantly enhanced the reproduction system, egg-laying ability, and embryo survivability. In conclusion, among the three policosanols, Cuban (Raydel®) policosanol had the strongest effect on survivability, improving dyslipidemia, liver protection, kidney, ovary, and testis with a restoration of the cell morphology, and the least ROS production and apoptosis-induced by HCD supplementation.

Anti-Inflammatory Activity of CIGB-258 against Acute Toxicity of Carboxymethyllysine in Paralyzed Zebrafish via Enhancement of High-Density Lipoproteins Stability and Functionality.

Background: Hyperinflammation is frequently associated with the chronic pain of autoimmune disease and the acute death of coronavirus disease (COVID-19) via a severe cytokine cascade. CIGB-258 (Jusvinza®), an altered peptide ligand with 3 kDa from heat shock protein 60 (HSP60), inhibits the systemic inflammation and cytokine storm, but the precise mechanism is still unknown. Objective: The protective effect of CIGB-258 against inflammatory stress of N-ε-carboxymethyllysine (CML) was tested to provide mechanistic insight. Methods: CIGB-258 was treated to high-density lipoproteins (HDL) and injected into zebrafish and its embryo to test a putative anti-inflammatory activity under presence of CML. Results: Treatment of CML (final 200 µM) caused remarkable glycation of HDL with severe aggregation of HDL particles to produce dysfunctional HDL, which is associated with a decrease in apolipoprotein A-I stability and lowered paraoxonase activity. Degradation of HDL3 by ferrous ions was attenuated by a co-treatment with CIGB-258 with a red-shift of the Trp fluorescence in HDL. A microinjection of CML (500 ng) into zebrafish embryos resulted in the highest embryo death rate, only 18% of survivability with developmental defects. However, co-injection of CIGB-258 (final 1 ng) caused the remarkable elevation of survivability around 58%, as well as normal developmental speed. An intraperitoneal injection of CML (final 250 µg) into adult zebrafish resulted acute paralysis, sudden death, and laying down on the bottom of the cage with no swimming ability via neurotoxicity and inflammation. However, a co-injection of CIGB-258 (1 µg) resulted in faster recovery of the swimming ability and higher survivability than CML alone injection. The CML alone group showed 49% survivability, while the CIGB-258 group showed 97% survivability (p < 0.001) with a remarkable decrease in hepatic inflammation up to 50%. A comparison of efficacy with CIGB-258, Infliximab (Remsima®), and Tocilizumab (Actemra®) showed that the CIGB-258 group exhibited faster recovery and swimming ability with higher survivability than those of the Infliximab group. The CIGB-258 group and Tocilizumab group showed the highest survivability, the lowest plasma total cholesterol and triglyceride level, and the infiltration of inflammatory cells, such as neutrophils in hepatic tissue. Conclusion: CIGB-258 ameliorated the acute neurotoxicity, paralysis, hyperinflammation, and death induced by CML, resulting in higher survivability in zebrafish and its embryos by enhancing the HDL structure and functionality.

Long-Term Alcohol Consumption Caused a Significant Decrease in Serum High-Density Lipoprotein (HDL)-Cholesterol and Apolipoprotein A-I with the Atherogenic Changes of HDL in Middle-Aged Korean Women.

Light-to-moderate alcohol drinking is associated with a low incidence of cardiovascular disease (CVD) via an elevation of high-density lipoproteins-cholesterol (HDL-C), particularly with the short-term supplementation of alcohol. However, there is no information on the change in the HDL qualities and functionalities between non-drinkers and mild drinkers in the long-term consumption of alcohol. This study analyzed the lipid and lipoprotein profiles of middle-aged Korean female non-drinkers, mild-drinkers, and binge-drinkers, who consumed alcohol for at least 10 years. Unexpectedly, the serum levels of HDL-C and apolipoprotein A-I (apoA-I) were decreased significantly depending on the alcohol amount; the binge-drinker group showed 18% and 13% lower HDL-C (p = 0.011) and apoA-I levels (p = 0.024), respectively, than the non-drinker group. Triglyceride (TG) and oxidized species, malondialdehyde (MDA), and low-density lipoproteins (LDL) levels were significantly elevated in the drinker groups. Interestingly, the binge-drinker group showed 1.4-fold higher (p = 0.020) cholesterol contents in HDL2 and 1.7-fold higher (p < 0.001) TG contents in HDL3 than those of the non-drinker group. The mild-drinker group also showed higher TG contents in HDL3 (p = 0.032) than the non-drinker group, while cholesterol contents were similar in the HDL3 of all groups. Transmission electron microscopy (TEM) showed that the non-drinker group showed a more distinct and clear particle shape of the LDL and HDL image with a larger particle size than the drinker group. Electrophoresis of LDL showed that the drinker group had faster electromobility with a higher smear band intensity and aggregation in the loading position than the non-drinker group. The HDL level of binge drinkers showed the lowest paraoxonase activity, the highest glycated extent, and the most smear band intensity of HDL and apoA-I, indicating that HDL quality and functionality were impaired by alcohol consumption. In conclusion, long-term alcohol consumption in middle-aged women, even in small amounts, caused a significant decrease in the serum HDL-C and apoA-I with atherogenic changes in LDL and HDL, such as an increase in TG and MDA content with a loss of paraoxonase activity.

Efficacy Comparison Study of Human Epidermal Growth Factor (EGF) between Heberprot-P® and Easyef® in Adult Zebrafish and Embryo under Presence or Absence Combination of Diabetic Condition and Hyperlipidemia to Mimic Elderly Patients.

Recombinant human epidermal growth factor (EGF) has been used to treat adult diabetic foot ulcers and pediatric burns by facilitating wound healing and epithelization, especially for elderly patients. Several formulation types of EGF from different expression hosts are clinically available, such as intralesional injection and topical application. On the other hand, no study has compared the in vivo efficacy of EGF products directly in terms of tissue regeneration and wound healing activity. The present study compared two commercial products, Heberprot-P75® and Easyef®, in terms of their tissue regeneration activity in adult zebrafish and the developmental speed of zebrafish embryos. Fluorescence spectroscopy showed that the two EGF products had different Trp fluorescence emission spectra: Easyef® showed a wavelength of maximum fluorescence (WMF) of 337 nm with weak fluorescence intensity (FI), while Heberprot-P75® showed WMF of 349 nm with a 4.1 times stronger FI than that of Easyef®. The WMF of Heberprot-P75® was quenched by adding singlet oxygen in ozonated oil, while the WMF of Easyef® was not quenched. Treatment with Heberprot-P75® induced greater embryo development speed with a higher survival rate after exposure to EGF in water and microinjection into embryos. Under normal diet (ND) consumption, Heberprot-P75® showed a 1.4 times higher tail fin regeneration activity than Easyef® during seven days from the intraperitoneal injection (10 µL, 50 µg/mL) after amputating the tail fin. Under ND consumption and diabetic condition caused by streptozotocin (STZ) treatment, Heberprot-P75® showed 2.1 times higher tail fin regeneration activity than Easyef® from the same injection and amputation protocol. Under a high-cholesterol diet (HCD) alone, Heberprot-P75® showed 1.2 times higher tail fin regeneration activity than the Easyef® group and PBS group from the same injection and amputation. Under diabetic conditions (STZ-injected) and HCD consumption, the Heberprot-P75® group showed 1.7 and 1.5 times higher tail fin regeneration activity than the Easyef® group and PBS group, respectively, with a distinct and clean regeneration pattern. In contrast, the Easyef® group and PBS group showed ambiguous regeneration patterns with a severe fissure of the tail fin, which is a typical symptom of a diabetic foot. In conclusion, Heberprot-P75® and Easyef® have different Trp fluorescence properties in terms of the WMF and fluorescence quenching. Treatment of Heberprot-P75® induced a greater developmental speed of zebrafish embryos in both water exposure and microinjection. Heberprot-P75® induced significantly higher wound healing and tissue regeneration activity than Easyef® and PBS in the presence or absence of diabetic conditions and cholesterol supplementation.

Ozonated Sunflower Oil Exerted Protective Effect for Embryo and Cell Survival via Potent Reduction Power and Antioxidant Activity in HDL with Strong Antimicrobial Activity.

Ozonated sunflower oil (OSO) has potent antimicrobial effects, making it useful for topical applications to treat various skin diseases. On the other hand, regarding mechanistic insight, the antioxidant activity and cytoprotective effects of OSO are relatively less known. The current study compared the antioxidant ability and protective ability of OSO on cells and embryos against oxidative stress, such as H2O2 and oxidized low-density lipoproteins (oxLDL), to investigate its potential applications for wound-healing and anti-infection. OSO showed potent radical scavenging activity and ferric ion reduction ability that was up to 35% and 42% stronger than sunflower oil (SO) as a control in a dose-dependent manner. Measurement of the wavelength-maximum fluorescence (WMF) of high-density lipoproteins (HDL) revealed different behavior between OSO and SO treatment (final 1-16%). The OSO treatment caused a 12 nm red shift of Trp movement from 345 nm (at 0%) to 357 nm (at 16%), while SO caused a 12 nm blue shift of Trp movement from 345 nm (at 0%) to 333 nm (at 16%). The fluorescence intensity of HDL3 was diminished remarkably by the OSO treatment by up to 80% from the initial level, while SO-treated HDL did not. OSO-treated HDL3 showed slower electromobility with stronger band intensity and bigger HDL particle sizes than those of SO-treated HDL3. The paraoxonase-1 (PON-1) activity of HDL3 was enhanced by a co-treatment of OSO that was up to 2.3 times higher than HDL3 alone in a dose-dependent manner, whereas the co-treatment of SO even inhibited the PON activity. The cell viability of RAW264.7 by the OSO treatment was 3.3 times higher than the SO treatment at a high dose range (from 10% to 50%, final). The OSO also exhibited more cytoprotective effects than SO in brain microglial cells in the presence of H2O2 (final 0.03%); treatment with OSO impeded apoptosis and reduced ROS production more than an SO treatment did. In the presence of H2O2 alone, 86 ± 5% of the embryos were killed by cell explosion after 24 h, but a co-treatment of OSO (final 4%) resulted in almost no embryo death (98% survivability). Injection of oxLDL (15 ng of protein) into zebrafish embryos caused acute death, while the co-injection of OSO (final 2%) resulted in 2.8 times higher survivability than oxLDL alone. These results suggest new effects of ozonated oil, such as enhanced antioxidant activity, more cytoprotective ability, and higher embryo protection against oxidative stress. These results may be useful in developing new methods for the quality control of ozonated oil and an assessment of its efficacy.

Short-term regulation of the hepatic activities of cytochrome P450 and glutathione S-transferase by nose-only cigarette smoke exposure in mice.

The present study aimed to determine the effects of cigarette smoke on the regulation of hepatic cytochrome P450 (CYP) and glutathione S-transferase (GST) enzymes in male BALB/c mice exposed to nose-only cigarette smoke for 4 days. There were no significant increases in serum liver injury markers (alanine aminotransferase and aspartate aminotransferase) or oxidative stress (total antioxidant capacity, malondialdehyde, and glutathione disulfide/reduced glutathione) following cigarette smoke exposure, but malondialdehyde was elevated in the bronchoalveolar lavage fluid of smoke-exposed mice. Additionally, the hepatic microsomal protein levels of Cyp1a and Cyp2b, and the activities of ethoxyresorufin O-deethylase, pentoxyresorufin O-depenylase, and chlorzoxazone 6-hydrxylase, were elevated in smoke-exposed mice. Interestingly, the hepatic activities of GST toward 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, and ethacrynic acid, but not cumene hydroperoxide were enhanced by cigarette smoke exposure, which was consistent with the increased expression levels of mu- and pi-class GSTs, but not alpha-class GSTs, observed in immunoblot analyses. These findings indicate that the short-term inhalation of cigarette smoke induces drug-metabolizing enzymes such as CYP1A, CYP2B, and mu/pi-class GSTs in the absence of hepatic injury and oxidative stress. Furthermore, smoking may alter hepatic drug metabolism, as well as the disposition and toxicity of xenobiotics, including some therapeutic drugs and cigarette smoke constituents.