P450scc Research Articles

Liver-specific Lxr inhibition represses reverse cholesterol transport in cholesterol-fed mice

Background and aimsHigh density lipoprotein (HDL) exerts an anti-atherosclerotic effect via reverse cholesterol transport (RCT). Several phases of RCT are transcriptionally controlled by Liver X receptors (Lxrs). Although macrophage Lxrs reportedly promote RCT, it is still uncertain whether hepatic Lxrs affect RCT in vivo. MethodsTo inhibit Lxr-dependent pathways in mouse livers, we performed hepatic overexpression of sulfotransferase family cytosolic 2B member 1 (Sult2b1) using adenoviral vector (Ad-Sult2b1). Ad-Sult2b1 or the control virus was intravenously injected into wild type mice and Lxrα/β double knockout mice, under a normal or high-cholesterol diet. A macrophage RCT assay and an HDL kinetic study were performed. ResultsHepatic Sult2b1 overexpression resulted in reduced expression of Lxr-target genes - ATP-binding cassette transporter G5/G8, cholesterol 7α hydroxylase and Lxrα itself - respectively reducing or increasing cholesterol levels in HDL and apolipoprotein B–containing lipoproteins (apoB-L). A macrophage RCT assay revealed that Sult2b1 overexpression inhibited fecal excretion of macrophage-derived 3H-cholesterol only under a high-cholesterol diet. In an HDL kinetic study, Ad-Sult2b1 promoted catabolism/hepatic uptake of HDL-derived cholesterol, thereby reducing fecal excretion. Finally, in Lxrα/β double knockout mice, hepatic Sult2b1 overexpression increased apoB-L levels, but there were no differences in HDL levels or RCT compared to the control, indicating that Sult2b1-mediated effects on HDL/RCT and apoB-L were distinct: the former was Lxr-dependent, but not the latter. ConclusionsHepatic Lxr inhibition negatively regulates circulating HDL levels and RCT by reducing Lxr-target gene expression.

Dan-shen Yin promotes bile acid metabolism and excretion to prevent atherosclerosis via activating FXR/BSEP signaling pathway

Ethnopharmacological relevanceDan-shen Yin (DSY), a traditional prescription, has been demonstrated to be effective in decreasing hyperlipidemia and preventing atherosclerosis (AS), but its mechanism remains unknown. We hypothesized that DSY activates farnesoid X receptor (FXR) to promote bile acid metabolism and excretion, thereby alleviating AS. Aim of the studyThis study was designed to explore whether DSY reduces liver lipid accumulation and prevents AS by activating FXR and increasing cholesterol metabolism and bile acid excretion. Materials and methodsThe comprehensive chemical characterization of DSY was analyzed by UHPLC-MS/MS. The AS models of ApoE−/− mice and SD rats was established by high-fat diet and high-fat diet combined with intraperitoneal injection of vitamin D3, respectively. The aortic plaque and pathological changes were used to evaluate AS. Lipid levels, H&E staining and oil red O staining were used to evaluate liver lipid accumulation. The cholesterol metabolism and bile acid excretion were evaluated by enzyme-linked immunosorbent assay, UPLC-QQQ/MS. In vitro, the lipid and FXR/bile salt export pump (BSEP) levels were evaluated by oil red O staining, real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. ResultsA total of 36 ingredients in DSY were identified by UPLC-MS/MS analysis. In vivo, high-dose DSY significantly inhibited aortic intimal thickening, improved arrangement disorder, tortuosity, and rupture of elastic fibers, decreased lipid levels, and reduced the number of fat vacuoles and lipid droplets in liver tissue in SD rats and ApoE−/− mice. Further studies found that high-dose DSY significantly reduced liver lipid and total bile acids levels, increased liver ursodeoxycholic acid (UDCA) and other non-conjugated bile acids levels, increased fecal total cholesterol (TC) levels, and augmented FXR, BSEP, cholesterol 7-alpha hydroxylase (CYP7A1), ATP binding cassette subfamily G5/G8 (ABCG5/8) expression levels, while decreasing ASBT expression levels. In vitro studies showed that DSY significantly reduced TC and TG levels, as well as lipid droplets, while also increasing the expression of ABCG5/8, FXR, and BSEP in both HepG2 and Nr1h4 knockdown HepG2 cells. ConclusionThis study demonstrated that DSY promotes bile acid metabolism and excretion to prevent AS by activating FXR. For the prevent of AS and drug discovery provided experimental basis.

Aromatic Amino Acids Promote Lipid Metabolism Disorders by Increasing Hepatic Bile Acid Synthesis

BackgroundObesity is a progressive metabolic disease that begins with lipid metabolism disorders. Aromatic amino acids (AAAs), including tryptophan, phenylalanine, and tyrosine, have diverse biological activities as nutrients. However, the underlying mechanisms by which AAAs affect lipid metabolism are unclear. ObjectivesThis study was designed to investigate the possible roles and underlying molecular mechanisms of AAA in the pathogenesis of lipid metabolism disorders. MethodsWe added an AAA mixture to the high-fat diet (HFD) of mice. Glucose tolerance test was recorded. Protein expression of hepatic bile acid (BA) synthase and mRNA expression of BA metabolism-related genes were determined. Hepatic BA profiles and gut microbial were also determined in mice. ResultsThe results showed that AAA significantly increased body weight and white adipose tissue, aggravated liver injury, impaired glucose tolerance and intestinal integrity, and significantly increased hepatic BA synthesis by inhibiting intestinal farnesoid X receptor (FXR). Moreover, AAA increased the content of total BA in the liver and altered the hepatic BA profile, with elevated levels of lithocholic acid, glycochenodeoxycholic acid, and glycoursodeoxycholic acid. AAA markedly increased the levels of proteins involved in BA synthesis (cholesterol 7α-hydroxylase and oxysterol 7α-hydroxylase) and inhibited the intestinal FXR. Gut microbial composition also changed, reducing the abundance of some beneficial bacteria, such as Parvibacter and Lactobacillus. ConclusionsUnder HFD conditions, AAAs stimulate BA synthesis in both the classical and alternative pathways, leading to aggravation of liver injury and fat deposition. Excessive intake of AAA disrupts BA metabolism and contributes to the development of lipid metabolism disorders, suggesting that AAA may be a causative agent of lipid metabolism disorders.

Mechanism of Tianma-Gouteng granules lowering blood pressure based on the bile acid-regulated Farnesoid X Receptor-Fibroblast Growth Factor 15- Cholesterol 7α-hydroxylase pathway

Ethnopharmacological relevanceTianma-Gouteng granules (TGG) is a traditional Chinese medicine (TCM) compound that was first recorded by modern medical practitioner Hu Guangci in "New Meaning of the Treatment of Miscellaneous Diseases in Traditional Chinese Medicine". It is widely used to treat hypertensive vertigo, headache and insomnia. Aim of studyTo investigate the antihypertensive effect of TGG and explore its mechanism. Materials and methodsSpontaneously hypertensive rats (SHR) were prepared a model of the ascendant hyperactivity of liver yang syndrome (AHLYS), blood pressure and general state of rats were recorded. A series of experiments were performed by enzyme-linked immunosorbent assay (ELISA), ultra high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS), 16S rRNA sequencing, real-time fluorescence quantitative PCR (RT-qPCR), and enzymatic colorimetry. ResultsTGG can effectively lower blood pressure and improve related symptoms. TGG significantly reduced the levels of IL-1β, IL-6, TNF-α, Renin and AngII. A total of 17 differential metabolites were found in plasma, with the two most potent metabolic pathways being glycerophospholipid metabolism and primary bile acid biosynthesis. After TGG intervention, 7 metabolite levels decreased and 10 metabolite levels increased. TGG significantly increased the relative abundance of Desulfovibio, Lachnoclostridium, Turicibacter, and decreased the relative abundance of Alluobaculum and Monoglobu. TGG also downregulated Farnesoid X Receptor (FXR) and Fibroblast Growth Factor 15 (FGF15) levels in the liver and ileum, upregulated Cholesterol 7α-hydroxylase (CYP7A1) levels, and regulated total bile acid (TBA) levels. ConclusionTGG can regulate bile acid metabolism through liver-gut axis, interfere with related intestinal flora and plasma metabolites, decrease blood pressure, and positively influence the pathologic process of SHR with AHLYS. When translating animal microbiota findings to humans, validation studies are essential to confirm reliability and applicability, particularly through empirical human research.

Effect of Day Length on Growth and Gonadal Development in Meishan Male Pigs.

Day length is a critical environmental factor for regulating animal growth and development. This study aimed to investigate the effects of different day lengths on the developmental changes of growth parameters, testicular sizes, testosterone secretion in Meishan male pigs, and steroidogenesis proteins and melatonin receptors. Fourteen Meishan male pigs (10 weeks (wks) of age) with the same parity, paired in litter and body weight (BW), were evenly allocated into a short-day-length group (SDL, 10 light/14 dark) and long-day-length group (LDL, 14 light/10 dark). After 12 wks of the experiment, the LDL-treated boars had more lying time and less exploring time. The LDL treatment led to significant increases in body height, chest circumference, testicular length, testicular weight, crude protein digestibility, and fecal testosterone at the 10th and 12th wks of the experiment, and cortisol at the 10th wk, compared to the SDL treatment, with no differences in the final BW, testicular width, and epididymis weight. Furthermore, the LDL treatment significantly increased the protein levels of melatonin receptor 1b (MT2), aromatase (CYP19), and steroidogenic factor 1 (SF1) in the testis, with no differences in the protein levels of melatonin receptor 1a (MT1), steroidogenic acute regulatory (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), and cholesterol side-chain cleavage enzyme (P450scc). The present study suggests that day length has an effect on the growth and gonadal development in male pigs maybe via MT2 and influences steroid synthesis and secretion in the testis. Therefore, proper day length should be considered in male pig breeding.

Involvement of Bile Acid Metabolism and Gut Microbiota in the Amelioration of Experimental Metabolism-Associated Fatty Liver Disease by Nobiletin.

Metabolism-associated fatty liver disease (MAFLD), a growing health problem worldwide, is one of the major risks for the development of cirrhosis and liver cancer. Oral administration of nobiletin (NOB), a natural citrus flavonoid, modulates the gut microbes and their metabolites in mice. In the present study, we established a mouse model of MAFLD by subjecting mice to a high-fat diet (HFD) for 12 weeks. Throughout this timeframe, NOB was administered to investigate its potential benefits on gut microbial balance and bile acid (BA) metabolism using various techniques, including 16S rRNA sequencing, targeted metabolomics of BA, and biological assays. NOB effectively slowed the progression of MAFLD by reducing serum lipid levels, blood glucose levels, LPS levels, and hepatic IL-1β and TNF-α levels. Furthermore, NOB reinstated diversity within the gut microbial community, increasing the population of bacteria that produce bile salt hydrolase (BSH) to enhance BA excretion. By exploring further, we found NOB downregulated hepatic expression of the farnesoid X receptor (FXR) and its associated small heterodimer partner (SHP), and it increased the expression of downstream enzymes, including cholesterol 7α-hydroxylase (CYP7A1) and cytochrome P450 27A1 (CYP27A1). This acceleration in cholesterol conversion within the liver contributes to mitigating MAFLD. The present findings underscore the significant role of NOB in regulating gut microbial balance and BA metabolism, revealing that long-term intake of NOB plays beneficial roles in the prevention or intervention of MAFLD.

Increased oxidative stress is associated with hyperandrogenemia in polycystic ovary syndrome evidenced by oxidized lipoproteins stimulating rat ovarian androgen synthesis in vitro.

To determine the relationship between serum total testosterone (TT) levels and oxidative stress indices in patients with polycystic ovary syndrome (PCOS), and to investigate the effect of oxidative stress on androgen synthesis and its mechanism in rat ovarian theca-interstitial (T-I) cells. Clinical, hormonal, metabolic, and oxidative stress parameters were analyzed in a cross-sectional case-control study including 626 patients with PCOS and 296 controls. The effects of oxidized low-density lipoprotein (ox-LDL) and oxidized high-density lipoprotein (ox-HDL) on cell proliferation, TT secretion, and expression of key enzymes involved in testosterone synthesis were evaluated in T-I cells. Serum TT levels were elevated with an increase in ox-LDL levels, whereas glutathione concentrations were lower in the high-TT subgroup than in the low-TT subgroup. The average ovarian volume and ox-LDL and malondialdehyde levels were significant predictors of TT levels in the multivariate regression models. In a rat ovarian T-I cell model, lipoprotein and oxidized lipoprotein treatments stimulated proliferation and promoted testosterone secretion. The mRNA and protein levels of 17α-hydroxylase were significantly higher in oxidized lipoprotein-treated cells than those in lipoprotein-treated cells. The mRNA levels of cholesterol side chain cleavage enzyme and steroidogenic acute regulatory protein were also significantly higher in ox-HDL-treated cells than in HDL-treated cells. Oxidative stress can promote androgen production by up-regulating the expression of testosterone synthesis-related enzymes in vitro and may be an essential factor in elevating serum TT levels in patients with PCOS.

Ginsenosides retard atherogenesis via remodelling host-microbiome metabolic homeostasis.

Panax ginseng is widely applied in the adjuvant treatment of cardiometabolic diseases in clinical practice without clear mechanisms. This study aims to clearly define the efficacy and underlying mechanism of P.ginseng and its active components in protecting against atherosclerosis. The anti-atherogenic efficacy of total ginseng saponin extract (TGS) and its components was evaluated on Ldlr-/- mice. Gut microbial structure was analysed by 16S rRNA sequencing and PCR. Bile acid profiles were revealed using targeted metabolomics with LC-MS/MS analysis. The contribution of gut microbiota to atherosclerosis was assessed by co-housing experiments. Ginsenoside Rb1, representing protopanaxadiol (PPD)-type saponins, increased intestinal Lactobacillus abundance, resulting in enhanced bile salt hydrolase (BSH) activity to promote intestinal conjugated bile acid hydrolysis and excretion, followed by suppression of enterohepatic farnesoid X receptor (FXR)-fibroblast growth factor 15 (FGF15) signal, and thereby increased cholesterol 7α-hydroxylase (CYP7A1) transcriptional expression and facilitated metabolic elimination of cholesterol. Synergistically, protopanaxatriol (PPT)-type saponins, represented by ginsenoside Rg1, protected against atherogenesis-triggered gut leak and metabolic endotoxaemia. Ginsenoside Rg1 directly induced mucin production to nutritionally maintain Akkermansia muciniphila, which reciprocally inhibited gut permeation. Rb1/Rg1 combination, rather than a single compound, can largely mimic the holistic efficacy of TGS in protecting Ldlr-/- mice from atherogenesis. Our study provides strong evidence supporting TGS and ginsenoside Rb1/Rg1 combinations as effective therapies against atherogenesis, via targeting different signal nodes by different components and may provide some elucidation of the holistic mode of herbal medicines.

Butorphanol inhibits androgen biosynthesis and metabolism in rat immature Leydig cells in vitro.

Butorphanol is a synthetic opioid analgesic medication that is primarily used for the management of pain. Butorphanol may have an inhibitory effect on androgen biosynthesis and metabolism in rat immature Leydig cells. The objective of this study was to investigate the influence of butorphanol on androgen secretion by rat Leydig cells isolated from the 35-day-old male rats. Rat Leydig cells were cultured with 0.5-50 μM butorphanol for 3h in vitro. Butorphanol at 5 and 50μM significantly inhibited androgen secretion in immature Leydig cells. At 50μM, butorphanol also blocked the effects of luteinizing hormone (LH) and 8bromo-cAMP-stimulated androgen secretion and 22R-hydroxycholesterol- and pregnenolone-mediated androgen production. Further analysis of the results showed that butorphanol downregulated the expression of genes involved in androgen production, including Lhcgr (LH receptor), Cyp11a1 (cholesterol side-chain cleavage enzyme), Srd5a1 (5α-reductase 1), and Akr1c14 (3α-hydroxysteroid dehydrogenase). Additionally, butorphanol directly inhibited HSD3B1 (3β-hydroxysteroid dehydrogenase 1) and SRD5A1 activity. In conclusion, butorphanol may have side effects of inhibiting androgen biosynthesis and metabolism in Leydig cells.

Testosterone synthesis was inhibited in the testis metabolomics of a depression mouse model

IntroductionDepression is a common emotional disorder. Previous studies have suggested that depression is associated with the central nervous system. Recent studies have suggested that reduced testosterone level is the core inducement of depression. Testis is the vital organ for the synthesis of testosterone. How does testis mediate depression is still unknown. ObjectivesWe adopted a classical depression model of mouse caused through chronic mild stress (CMS). The metabolomics liquid chromatography-mass spectrometry was adopted to analyse the influence of CMS on testis metabolism. Then we confirmed the possible abnormal metabolism of the testis in depression mice by pathway analysis and molecular biological technique. ResultsCompared with control mice, 16 differential metabolites were found in CMS mice by multivariate statistical analysis. In comparison with control mice, CMS mice showed higher levels for campesterol, ribitol, citric acid, platelet activating factor, guanosine, cytosine and xanthine and lower levels for docosahexaenoic acid, hippuric acid, creatine, testosterone, dehydroepiandrosterone, progesterone, l-carnitine, acetyl carnitine and propionyl carnitine. The pathway analysis indicated that these differential metabolites are associated with steroid hormone synthesis, purine metabolism and phenylalanine metabolism. In addition, we also first discovered that testicular morphology in depression mice was damaged and steroid hormone synthetases (including steroidogenic acute regulatory protein and P450 cholesterol side chain cleavage) were inhibited. ConclusionThese findings may be helpful to parse molecular mechanisms of pathophysiology of depression. It also pointed out the direction to search for potential therapy schedules for male depression and provide novel insights into exploring the pathogenesis of male depression.

Prevention of high-fat-diet-induced obesity in mice by soluble dietary fiber from fermented and unfermented millet bran

Obesity-related diabetes, cardiovascular disease, and hypertension pose many risks to human health. Thus, mice on a high-fat diet were gavaged with millet bran (unfermented/fermented) soluble dietary fiber (RSDF/FSDF, 500 mg·kg−1) for 10 weeks in current research, and then evaluated the various biological indicators. These findings revealed that RSDF and FSDF supplements could prevent fat synthesis by inhibiting sterol regulatory element-binding protein-1c gene expression. The RSDF supplements can also accelerate fat catabolism through enhanced the mRNA expression levels of adipose triglyceride lipase and peroxisome proliferator-activated receptor α. FSDF supplements can prevent obesity by decreasing 3-hydroxy-3-methyl-glutaryl-CoA reductase expression and increasing cholesterol 7α-hydroxylase expression. Moreover, FSDF also controls obesity development by lowering total cholesterol and low-density lipoprotein cholesterol levels in the blood, triglyceride, total cholesterol, and bile acid levels in the liver. Notably, FSDF supplements can promote Bacteroides and Prevotella propagation; excretive propionic acid binds to free fatty acid receptor 2/3 and then stimulates intestinal epithelial cells to generate glucagon-like-peptide-1 and peptide YY, which can reduce food and energy intake and ultimately prevent obesity. All evidence suggests that FSDF supplements play a crucial role in preventing obesity.

Fluctuating and Stable High Temperatures Differentially Affect Reproductive Endocrinology of Female Pupfish.

For many fishes, reproductive function is thermally constrained such that exposure to temperatures above some upper threshold has detrimental effects on gametic development and maturation, spawning frequency, and mating behavior. Such impairment of reproductive performance at elevated temperatures involves changes to hypothalamic-pituitary-gonadal (HPG) axis signaling and diminished gonadal steroidogenesis. However, how HPG pathways respond to consistently high versus temporally elevated temperatures is not clear. Here, sexually mature Amargosa River Pupfish (Cyprinodon nevadensis amargosae) were maintained under thermal regimes of either stable ∼25°C (low temperature), diurnal cycling temperatures between ∼27 and 35°C (fluctuating temperature), or stable ∼35°C (high temperature) conditions for 50 days to examine effects of these conditions on HPG endocrine signaling components in the pituitary gland and gonad, ovarian and testicular gametogenesis status, and liver gene expression relating to oogenesis. Female pupfish maintained under stable high and fluctuating temperature treatments showed reduced gonadosomatic index values as well as a lower proportion of oocytes in the lipid droplet and vitellogenic stages. Females in both fluctuating and stable 35°C conditions exhibited reduced ovarian mRNAs for steroid acute regulatory protein (star), cholesterol side chain-cleavage enzyme, P450scc (cyp11a1), and 3β-hydroxysteroid dehydrogenase (3bhsd), while ovarian transcripts encoding 11β-hydroxysteroid dehydrogenase (11bhsd) and sex hormone-binding globulin (shbg) were elevated in females at constant 35°C only. Ovarian aromatase (cyp19a1a) mRNA levels were unaffected, but circulating 17β-estradiol (E2) was lower in females at 35°C compared to the fluctuating temperature condition. In the liver, mRNA levels for choriogenins and vitellogenin were downregulated in both the fluctuating and 35°C conditions, while hepatic estrogen receptor 2a (esr2a) and shbg mRNAs were elevated in 35°C females. Taken together, these results demonstrate the potential for elevated temperatures to impair ovarian steroidogenesis and reduce egg envelope and vitellogenin protein production in female C. n. amargosae pupfish, while also shedding light on how thermal regimes that only intermittently reach the upper thermal range for reproduction have differential impacts on reproductive endocrine pathways than constantly warm conditions.

Hepatotropic activity of a betulonic acid based compound.

Using the model of cyclophosphamide (CP)-induced immunosuppression in C57BL/6 mice, the hepatotropic effects of a conjugate of betulonic acid with 9-(4-methylpiperazin-1-ylmethyl)-2-(1,2,3-triazolyl) oreozelone (BABC) have been studied. In the liver of treated animals the expression of genes for cytochromes (CYP 1A1, CYP 1A2, CYP 3A44, CYP 2B10, CYP 2C29, CYP 17A1), PPARA, and cytokines (TNF-α, IL-1β, IL-12α, IL-10) and the relative levels of NF-κB p65, GST-π, and NAT-1 proteins were determined. On day six after administration of the compound and CP to animals a significant (3.2-fold) increase in the expression of the CYP 2B10 as compared to the control group was observed. Treatment of mice with the compound and CP also caused a 2.4-fold increase in the mRNA level of the pro-inflammatory TNF-α gene as compared to the group of animals receiving CP. Administration of the studied compound to intact animals was accompanied by a 2.5-fold increase in the IL-1β expression and a 1.8-fold decrease in the IL-10 expression as compared to the control group. An increase in the expression of pro-inflammatory cytokine genes in the liver of animals treated with the compound was accompanied by an increase in the content of NF-κB p65 (by 1.6 times), as well as an increase in the relative amount of NAT-1 protein (by 2.7 times) as compared to control animals.

Endogenous biosynthesis of docosahexaenoic acid (DHA) regulates fish oocyte maturation by promoting pregnenolone production.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), particularly docosahexaenoic acid (22:6n-3, DHA), play crucial roles in the reproductive health of vertebrates, including humans. Nevertheless, the underlying mechanism related to this phenomenon remains largely unknown. In this study, we employed two zebrafish genetic models, i.e., elovl2 -/- mutant as an endogenous DHA-deficient model and fat1 (omega-3 desaturase encoding gene) transgenic zebrafish as an endogenous DHA-rich model, to investigate the effects of DHA on oocyte maturation and quality. Results show that the elovl2 -/- mutants had much lower fecundity and poorer oocyte quality than the wild-type controls, while the fat1 zebrafish had higher fecundity and better oocyte quality than wild-type controls. DHA deficiency in elovl2 -/- embryos led to defects in egg activation, poor microtubule stability, and reduced pregnenolone levels. Further study revealed that DHA promoted pregnenolone synthesis by enhancing transcription of cyp11a1, which encodes the cholesterol side-chain cleavage enzyme, thereby stabilizing microtubule assembly during oogenesis. In turn, the hypothalamic-pituitary-gonadal axis was enhanced by DHA. In conclusion, using two unique genetic models, our findings demonstrate that endogenously synthesized DHA promotes oocyte maturation and quality by promoting pregnenolone production via transcriptional regulation of cyp11a1.

Cholesterol lowering in diet-induced hypercholesterolemic mice using Lactobacillus bile salt hydrolases with different substrate specificities.

The cholesterol-lowering effect of lactic acid bacteria with high activity of bile salt hydrolase (BSH) is unclear. We believe that distinguishing BSH substrate specificity is necessary to study the effect of various BSH enzymes. We engineered a BSH mutant enzyme recombinant strain named F67A, which exclusively hydrolyzes taurocholic acid (TCA) using site-directed mutagenesis, and a previously lab-constructed BSH recombinant strain, YB81 that exclusively hydrolyzes glycocholic acid (GCA). We also constructed the recombinant strain named NB5462, which carries the empty pSIP411 plasmid and was used as a blank control strain. The intestinal flora in pseudo-germ-free (PGF) mice in which intestinal flora were eliminated via antibiotics, and F67A successfully reduced serum cholesterol levels in high-cholesterol diet-fed mice, whereas YB81 did not yield the same results. However, YB81 regained its cholesterol-lowering capacity in specific pathogen-free (SPF) mice with intact intestinal flora. The cholesterol-lowering mechanism of F67A involved modifying the bile acid pool through BSH enzyme activity. This adjustment regulated the expression of intestinal farnesoid X receptor and subsequently elevated hepatic cholesterol 7α-hydroxylase (CYP7A1), effectively reducing cholesterol levels. Conversely, GCA, the substrate of YB81, was found in minimal quantities in mice, preventing it from inducing changes in bile acid pools. In the presence of intestinal flora, the YB81 BSH enzyme induced notable alterations in bile acids by regulating changes in the intestinal flora and BSH within the flora, ultimately resulting in cholesterol reduction. This is the first study investigating the substrate specificity of BSH, demonstrating that different substrate-specific BSH enzymes exhibit cholesterol-lowering properties. Additionally, we elaborate on the mechanism of BSH-mediated enterohepatic axis regulation.

Lingguizhugan oral solution alleviates MASLD by regulating bile acids metabolism and the gut microbiota through activating FXR/TGR5 signaling pathways.

The preservation of the Lingguizhugan (LGZG) decoction and patient compliance issue often limit the treatment of metabolic dysfunction-associated steatotic liver disease (MASLD). Hence, herein, an LGZG oral solution was developed for alleviating MASLD. Additionally, the potential mechanisms underlying LGZG-mediated MASLD mitigation were explored. A MASLD mouse model was constructed using oleic and palmitic acid-induced LO2 cells and a high-fat diet. The apoptosis, lipid deposition, and mouse liver function were analyzed to assess the therapeutic effects of the LGZG oral solution on MASLD. Serum untargeted metabolomics, gut microbiota, bile acid (BA) metabolism, immunohistochemistry, and Western blotting analyses were performed to investigate the potential mechanism of action of LGZG oral solution on MASLD. The LGZG oral solution ameliorated lipid deposition, oxidative stress, inflammation, and pathological damage. Serum untargeted metabolomics results revealed the LGZG-mediated regulation of the primary BA biosynthetic pathway. The 16S ribosomal RNA sequencing of the fecal microbiota showed that LGZG oral solution increased the relative abundance of the BA metabolism-associated Bacteroides, Akkermansia, and decreased that of Lactobacillus. Additionally, the BA metabolism analysis results revealed a decrease in the total taurine-α/β-muricholic acid levels, whereas those of deoxycholic acid were increased, which activated specific receptors in the liver and ileum, including farnesoid X receptor (FXR) and takeda G protein-coupled receptor 5 (TGR5). Activation of FXR resulted in an increase in short heterodimer partner and subsequent inhibition of cholesterol 7α-hydroxylase and sterol regulatory element-binding protein-1c expression, and activation of FXR also results in the upregulation of fibroblast growth factor 15/19 expression, and consequently inhibition of cholesterol 7α-hydroxylase, which correlated with hepatic BA synthesis and lipogenesis, ultimately attenuating lipid deposition and bile acid stasis, thereby improving MASLD. Altogether, the findings of this study suggest that modulating microbiota-BA-FXR/TGR5 signaling pathway may be a potential mechanism of action of LGZG oral solution for the treatment of MASLD.

Reduced oxygen concentrations regulate the phenotype and function of human granulosa cells in vitro and cause a diminished steroidogenic but increased inflammatory cellular reaction.

Oxygen (O2) concentrations have recently been discussed as important regulators of ovarian cells. Human IVF-derived granulosa cells (human GCs) can be maintained in vitro and are a widely used cellular model for the human ovary. Typically, GCs are cultured at atmospheric O2 levels (approximately around 20%), yet the O2 conditions in vivo, especially in the preovulatory follicle, are estimated to be much lower. Therefore, we comprehensively evaluated the consequences of atmospheric versus hypoxic (1% O2) conditions for 4 days on human GCs. We found lower cellular RNA and protein levels but unchanged cell numbers at 1% O2, indicating reduced transcriptional and/or translational activity. A proteomic analysis showed that 391 proteins were indeed decreased, yet 133 proteins were increased under hypoxic conditions. According to gene ontology (GO) enrichment analysis, pathways associated with metabolic processes, for example amino acid-catabolic-processes, mitochondrial protein biosynthesis, and steroid biosynthesis, were downregulated. Pathways associated with glycolysis, chemical homeostasis, cellular response to hypoxia, and actin filament bundle assembly were upregulated. In accordance with lower CYP11A1 (a cholesterol side-chain cleavage enzyme) levels, progesterone release was decreased. A proteome profiler, as well as IL-6 and IL-8 ELISA assays, revealed that hypoxia led to increased secretion of pro-inflammatory and angiogenic factors. Immunofluorescence studies showed nuclear localization of hypoxia-inducible factor 1α (HIF1α) in human GCs upon acute (2 h) exposure to 1% O2 but not in cells exposed to 1% O2 for 4 days. Hence, the role of HIF1α may be restricted to initiation of the hypoxic response in human GCs. The results provide a detailed picture of hypoxia-induced phenotypic changes in human GCs and reveal that chronically low O2 conditions inhibit the steroidogenic but promote the inflammatory phenotype of these cells.

Rare forms of congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders due to pathogenic variants in genes encoding enzymes and cofactors involved in adrenal steroidogenesis. Although 21-hydroxylase, 11β-hydroxylase, 3β-hydroxysteroid dehydrogenase type 2, 17α-hydroxylase/17,20-lyase, P450 oxidoreductase, steroidogenic acute regulatory protein, cholesterol side-chain cleavage enzyme deficiencies are considered within the definition of CAH, the term 'CAH' is often used to refer to '21-hydroxylase deficiency (21OHD)' since 21OHD accounts for approximately 95% of CAH in most populations. The prevalence of the rare forms of CAH varies according to ethnicity and geographical location. In most cases, the biochemical fingerprint of impaired steroidogenesis points to the specific subtypes of CAH, and genetic testing is usually required to confirm the diagnosis. Despite there are significant variations in clinical characteristics and management, most data about the rare CAH forms are extrapolated from 21OHD. This review article aims to collate the currently available data about the diagnosis and the management of rare forms of CAH.

The anti-hyperlipidemic effects of Poria cocos (Schw.) Wolf extract: Modulating cholesterol homeostasis in hepatocytes via PPARα pathway

Ethnopharmacological relevancePoria cocos (Schw.) Wolf (Polyporaceae, P.cocos), which is born on the pine root, has a history of more than two thousand years of medicine in China. P.cocos was first recorded in the Shennong's Herbal Classic, studies have proved its lipid-lowering effect. Aim of studyThe aim of study was to investigate the underlying mechanism of P.cocos extract on hyperlipidemia. Materials and methodsMale Sprague-Dawley (SD) rats aged 9–12 weeks were intraperitoneally (IP) injected with Triton-WR 1339 to establish an acute hyperlipidemia model. At 0 h and 20 h after the model was established, low and high doses of P.cocos extract or simvastatin were given twice. After 48 h, the rats were sacrificed, and liver and serum samples were collected for analysis. The cell model was constructed by treating L02 cells with 1% fat emulsion-10% FBS–RPMI 1640 medium for 48 h. At the same time, low and high doses of P.cocos extract and simvastatin were administered. Oil red O staining was used to evaluate the lipid accumulation in the cells, and H&E staining was used to evaluate the liver lesions of rats. Real-time quantitative PCR and western blotting were used to detect the expressions of lipid metabolism-related genes. ResultsP.cocos extract relieved lipid accumulation in vitro and alleviated hyperlipidemia in vivo. Both gene and protein expressions of peroxisome proliferator-activated receptor α (PPARα) were shown to be up-regulated by P.cocos extract. Additionally, P.cocos extract down-regulated the expressions of fatty acid synthesis-related genes sterol regulatory element-binding protein-1 (SREBP-1), Acetyl-CoA Carboxylase 1 (ACC1) and fatty acid synthase (FAS), while up-regulated the expressions of cholesterol metabolism-related genes liver X receptor-α (LXRα), ATP-binding cassette transporter A1 (ABCA1), cholesterol 7alpha-hydroxylase (CYP7A1) and low density lipoprotein receptor (LDLR), which were reversed by the treatment with the PPARα inhibitor GW6471. ConclusionP.cocos extract ameliorates hyperlipidemia and lipid accumulation by regulating cholesterol homeostasis in hepatocytes through PPARα pathway. This study provides evidence that supplementation with P.cocos extract could be a potential strategy for the treatment of hyperlipidemia.

Effect of Notch Signal Pathway on Steroid Synthesis Enzymes in TM3 Cells.

Studies have indicated that the conservative Notch pathway contributes to steroid hormone synthesis in the ovaries; however, its role in hormone synthesis of the testis remains unclear. We have previously reported Notch 1, 2, and 3 to be expressed in murine Leydig cells and that inhibition of Notch signaling caused G0/G1 arrest in TM3 Leydig cells. In this study, we have further explored the effect of different Notch signal pathways on key steroidogenic enzymes in murine Leydig cells. TM3 cells were treated with Notch signaling pathway inhibitor MK-0752, and different Notch receptors were also overexpressed in TM3 cells. We evaluated the expression of key enzymes of steroid synthesis, including p450 cholesterol side-chain cleavage enzyme (P450Scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and steroidogenic acute regulatory protein (StAR), and key transcriptional factors for steroid synthesis, including steroidogenic factor 1 (SF1), GATA-binding protein 4 (GATA4) and GATA6. We found the level of P450Scc, 3β-HSD, StAR and SF1 to be decreased after treatment with MK-0752, while overexpression of Notch1 up-regulated the expression of 3β-HSD, P450Scc, StAR and SF1. MK-0752 and overexpression of different Notch members had no influence on the expression of GATA4 and GATA6. In conclusion, Notch1 signaling may contribute to the steroid synthesis in Leydig cells through regulating SF1 and downstream steroidogenic enzymes (3β-HSD, StAR and P450Scc).