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4 Most Liver Damaging Supplements (Avoid Over Usage)Citrus aurantium for liver support -
Paraffin sections of zebrafish larvae were deparaffinized with xylene, rehydrated with ethanol, and sodium citrate was boiled for 10 min to recover the antigen, and cooled to room temperature. The next day, paraffin sections were washed three times with PBS, then incubated with goat anti-rabbit antibody for 2 h at room temperature.
The sections were washed with PBS, followed by stained with DAB for less than 10 min and terminated with ice water. The samples were counter-stained with hematoxylin, dehydrated, and finally sealed with a neutral glue.
The stained sections were photographed with an optical microscope Nikon Eclipse Ni-U; Nikon, Tokyo, Japan. The sections and cell slides were incubated with rabbit antibody diluted with 2.
After being washed with PBS, the samples were incubated with goat anti-rabbit antibody for 1 h at room temperature.
Then washed with PBS and incubated with DAPI for 10 min in the dark. The samples were washed three times with PBS, followed by sealed with anti-fluorescence quencher and photographed with an optical microscope Nikon Eclipse Ni-U; Nikon, Tokyo, Japan.
The ordered primers for each gene were obtained from BGI Tech Solutions Beijing Liuhe Co. and were showed in Table 1. Collected LO2 cells were washed three times with PBS, and lysed by cell lysate containing phosphatase inhibitor Sigma , RIPA lysis buffer Sigma and protease inhibitor Sigma for 15 min, centrifuged, and then aspirated the supernatant for protein quantification and protein denaturation.
A total of 30 μg protein was used for western blotting. All statistical analysis was performed with GraphPad Prism Version 8. The numerical results are expressed as mean ± standard deviation SD. We used the survival rate, the heart rate, the body length and morphological changes to observe the toxicology of limonin in zebrafish larvae.
Zebrafish larvae 3 dpf were treated with limonin for 72 h. Zebrafish larvae had no morphological abnormalities were observed at the tested concentrations of limonin Figure 2A , indicating that limonin has low drug toxicity to the morphology of zebrafish during development.
In the heart rate test and the body length test, different concentrations of limonin had no significant effect on the heart rate and body length of zebrafish larvae Figures 2C,D. According to the results of drug toxicity studies, we found that limonin was safe and had low toxicity, and FIGURE 2.
Toxicology of limonin in zebrafish larvae. The data are shown as mean ± SD. Zebrafish larvae were stimulated with 0. Based on this model, the protective effect of limonin on NAFLD was evaluated.
Hepatic steatosis is the main pathological feature in the early stage of NAFLD. The results showed that Based on the above results, the concentration of 50 μM was used to further verify the pharmacological effects of limonin on NAFLD.
Frozen sections of zebrafish were then stained with Oil Red O Figures 4A,C and Nile Red Figures 4B,D to further confirm that 50 μM limonin effectively reduced lipid accumulation in the liver after TAA exposure.
Fatty acid synthase FASN plays a key role in the initial process of lipid synthesis Angeles and Hudkins, QPCR results showed that the expression level of FASN mRNA in the TAA exposure group was increased, and decreased after being treated with 50 μM limonin Figure 4E. These data showed that limonin had the effect of ameliorating TAA-induced hepatic steatosis.
FIGURE 3. Limonin alleviated TAA-induced liver hepatic steatosis in zebrafish larvae. B Whole fish staining with Oil Red O. C Quantitative analysis of the liver gray value of Oil Red O using ImageJ software.
Data are shown as the mean ± SD. FIGURE 4. Limonin reduced lipid accumulation caused by TAA in zebrafish liver. A Oil red O staining of frozen sections of zebrafish larvae.
B Frozen liver sections of zebrafish larvae with liver-specific eGFP expression were stained with Nile Red. Figures are magnified as × C Quantitative analysis of the area of lipid droplet in liver based on Oil Red O staining.
D Quantitative analysis of the liver mean fluorescence intensity of Nile Red. E Real-time PCR analysis of FASN mRNA levels in zebrafish larvae. The mRNA levels were normalized to β-actin mRNA levels and presented as fold change compared with the control group.
We also verified the effect of limonin on lipid accumulation in hepatocytes in vitro. We first established an NAFLD model of LO2 cells using lipid mixture to study the effect of limonin on lipid accumulation in hepatocytes.
Based on the result of the cell viability experiment Figure 5A , we chose 10, 20, and 40 μM limonin for the experiment. Consistent with the results of the zebrafish experiments in vivo , both oil red O staining Figures 5D,E and Nile red staining Figures 5F,G showed that lipid mixture stimulation significantly induced intracellular lipid accumulation, while effectively reduced in limonin-treated groups.
Moreover, sterol regulatory element binding protein 1 SREBP-1 is a membrane-bound transcription factor that participates in many functions of lipid homeostasis, especially the process of lipid synthesis Guo et al.
Results of western blot showed that the expression levels of FASN and SREBP1 in the limonin-treated group were lower than model group Figures 5B,C. These results indicated that limonin also effectively reduced lipid accumulation in hepatocytes in vitro.
FIGURE 5. Limonin reduced lipid accumulation in LO2 cells stimulated by lipid mixture. B Western blot analysis of the expression of FASN and SREBP1.
GAPDH was used as an internal control. C Relative quantitative protein expression of FASN and SREBP1. D LO2 cells oil red O staining. E Quantitative analysis of the area of lipid droplets based on Oil Red O staining in LO2 cells.
F Nile red staining of LO2 cells. G Quantitative analysis of the mean fluorescence intensity of Nile Red. Data are shown as the mean ± SD from three independent experiments.
A large number of experimental and clinical data support that inflammatory cells, especially macrophages, play a central role in the occurrence and development of NAFLD and NASH Koyama and Brenner, And limonin has been reported to serve the effect of modulating immune and inflammatory responses to alleviate injury Ishak et al.
Therefore, we utilize zebrafish with transgenic strain macrophages-specific dsred-labeled to observe the distribution of macrophages in real time. We found that TAA exposure caused macrophages infiltration in liver, and 50 μM limonin could reduce the migration of macrophages to the liver Figures 6A,B.
Real-time PCR analysis also showed that compared with the control group, the TAA-treated group significantly increased the mRNA expression of Interleukin-6 IL-6 and Interleukin-1β IL-1β. However, these pro-inflammatory factors reduced in the limonin treatment group Figures 6E,H.
These results indicated that limonin reduced the infiltration of macrophages and the expression of inflammatory factors TNF-α, IL-1β and IL-6 secreted by macrophages in the liver, thereby ameliorating the inflammatory damage in the process of NAFLD.
FIGURE 6. Limonin reduced liver inflammatory damage caused by TAA. A Limonin reduced the infiltration of macrophages in the liver of zebrafish during TAA exposure.
B Relative expression of mpeg1 in zebrafish liver. E,H Real-time PCR analysis of IL-1β and IL-6 mRNA levels in zebrafish larvae. Free fatty acids, which are elevated in the liver by lipid accumulation or insulin resistance, lead to incomplete oxidation in the mitochondria, peroxisomes, and microsomes, leading to the production of ROS, and subsequently accelerates the progression of NAFLD Chen et al.
Elevated levels of ROS modulate the activity of immune signaling, induce DNA damage and affect the expression and activity of key enzymes involved in lipid metabolism Srinivas et al.
GSH is a vital factor in the endogenous protection system to eliminate ROS in NAFLD Brigelius Flohe and Maiorino, Therefore, DHE and NA-8 fluorescent probes were used to detect the distribution and levels of ROS and GSH to evaluate the TAA-induced oxidative damage in the liver of zebrafish larvae and the protective effect of limonin.
The results showed strong red fluorescence appeared in zebrafish liver after TAA exposure, and 50 μM limonin could effectively reduce TAA-induced ROS accumulation Figures 7A,C. Simultaneously, the blue fluorescence emitted by the NA-8 probe in the zebrafish liver of TAA-treated group was weakened, indicating glutathione depletion caused by TAA.
However, glutathione in the liver increased after limonin treatment, which also strongly supported the protective effect of limonin on oxidative stress Figures 7B,D. Besides, real-time PCR analysis showed that compared with the control group, the mRNA level of antioxidant gene gpx1a in model group was significantly reduced, but reversed after 50 μM limonin treatment Figure 7E.
These outcomes demonstrated that limonin played an important role in resisting oxidative stress in the progression of NAFLD induced by TAA. FIGURE 7. Limonin protected zebrafish larvae against oxidative stress. A Fluorescence micrographs of DHE. Figures are magnified as 50×.
B Fluorescence micrographs of NA Figures are magnified as ×50 C ROS quantification according to the mean intensity of red fluorescence. D GSH quantification according to the mean intensity of blue fluorescence.
E Real-time PCR analysis of gpx1a mRNA levels in zebrafish larvae. Transcription factor nuclear factor-erythroid 2-related factor 2 NRF2 controls cellular adaptation to oxidants and electrophiles by inducing antioxidant genes like heme oxygenase-1 HO-1 in response to redox stress Hayes and Mcmahon, ; Villeneuve et al.
In our study, immunofluorescence staining and real-time quantitative PCR showed that compared with the control group, the expression of NRF2 and HO-1 proteins were down-regulated after TAA exposure, whereas increased with limonin treatment Figures 8A—E.
Meanwhile, we also detected the distribution of NRF2 in LO2 cells. NRF2 was mainly located in the cytoplasm in normal cells, and lipid mixture could partially transfer NRF2 from the cytoplasm to the nucleus.
Moreover, limonin treatment significantly promoted the nuclear expression of Nrf2 Figures 8J,K. Overall, these results supported the important role of limonin-mediated antioxidant capacity in the development of NAFLD. FIGURE 8. C—D Quantitative analysis of the liver mean fluorescence intensity of NRF2 and HO E Real-time PCR analysis of NRF2 mRNA levels in zebrafish larvae.
F Western blot analysis of the expression of HO-1 and NRF2. G Relative quantitative protein expression of NRF2 and HO H Immunofluorescence staining of HO-1 in LO2 cells. I Cellular fluorescence intensity of HO J Immunofluorescence staining of NRF2 in LO2 cells.
K The respective fluorescence intensity of the nucleus, cytoplasm and ratio of NRF2. As the rising prevalence has caused an increasing economic burden, and with more and more patients with liver cirrhosis and end-stage liver disease requiring liver transplantation, NAFLD has become a disease of major concern Friedman et al.
However, the pathogenesis of NAFLD has not yet been fully elucidated, and the determination of therapeutic targets and the advancement of drug development are severely limited. Current research shows that limonin has a wide range of pharmacological effects, including anti-cancer, anti-inflammation, anti-bacterial, anti-viral, anti-oxidation and liver protection properties Fan et al.
But the therapeutic potential of limonin in NAFLD is still unknown. In this study, we clarified that limonin could alleviate fatty liver disease in vitro and in vivo experiments, including decreased liver lipid accumulation and down-regulated the expression of lipogenic transcription factors FASN and SREBP1, which were up-regulated by de novo lipogenesis and ultimately induce steatosis Anderson and Borlak, It is believed that various subsequent injuries such as inflammatory infiltration and oxidative stress can accelerate the development of NAFLD.
And obesity and cytokine factors have been shown to be linked by certain stimulating factors like stem cell growth factor-beta Tarantino et al. In this study, we used macrophage-labeled zebrafish to observe the distribution of macrophages in real time and found that limonin reduced TAA-induced recruitment of macrophages in the liver.
It can also interact with certain medications, such as blood thinners, and can cause dangerous interactions. It is important to speak with a doctor before taking any dietary supplement, including Neoeriocitrin, to ensure it is safe for you to take.
Vitamin C tablets, Neoeriocitrin capsules, Neoeriocitrin powder, Neoeriocitrin chewable tablets, Neoeriocitrin liquid drops. Neoeriocitrin is not a dietary supplement that is regulated across the world.
However, it is regulated in some countries, such as the United States, where it is classified as a dietary ingredient and is subject to the regulations of the Dietary Supplement Health and Education Act DSHEA.
In other countries, such as the European Union, Neoeriocitrin is not regulated as a dietary supplement, but is instead regulated as a food additive. Tags: Vitamins. Get the most up-to-date information about Neoeriocitrin with SGS Digicomply. Stay informed about current regulations, incident monitoring, risk prevention, scientific insights, mentions in the media, and relevant updates.
Smart GPT-like search, customizable dashboard, and comprehensive guides tailored to your specific area, product, and target market. Dietary Supplements Database Neoeriocitrin November 3 Where is Neoeriocitrin used? How is Neoeriocitrin used in the food industry? Prevention by chlorpromazine of an accelerated phospholipid degradation and associated membrane dysfunction.
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Bitter orange Suppor aurantiumalso known as qurantium orange and Seville orange, is a citrus Cittrus with a multitude of Natural hunger reduction. This article liveg all you need to know about Citrus aurantium for liver support orange, including its role in weight loss and skin health, as well as its overall safety as a supplement. The bitter orange plant thrives in subtropical regions but can withstand adverse environmental conditions like frost for short periods 2. Oval or oblong in shape, the fruit is red-orange when ripe and has a distinctively thick, dimpled skin. There are 23 cultivars of the fruit, the most prominent of which is Bergamot.
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