Browse

Hypothyroidism is a common endocrine disease caused by a deficiency of thyroid hormones, which could affect the hypothalamus–pituitary–gonadal (HPG) axis and cause additional severe fertility problems. However, the pathogenesis of abnormal reproductive capacity caused by hypothyroidism and whether there are differences between females and males need more study. Here, we constructed a prolonged neonatal hypothyroid rat model using 6-propyl-2-thiouracil (PTU). H&E staining and RNA-sequencing were performed to detect histopathological and transcriptome changes. Our results indicated that the numbers of ventromedial hypothalamus nuclei were increased, and the number of pituitary chromophobes was sharply increased, whereas the proportion of pituitary acidophils and pituitary basophils were obviously reduced. The differentially expressed genes of the HPG axis organs were identified, and different tissues shared similar steroid hormone and oxidative stress-related terms in gene ontology analysis. Weighted gene co-expression network analysis (WGCNA) and differential expression analysis indicated oxidative stress, and apoptosis-related genes were more enriched in male hypothyroid pituitaries, whereas the serum levels of growth hormone, follicle-stimulating hormone, and luteinizing hormone that were detected by ELISA were also reduced more in male hypothyroid rats, suggesting that prolonged neonatal hypothyroidism may have a more significant impact on male pituitaries. Moreover, the multi-organ oxidative stress in hypothyroid rats was confirmed by the higher expression of oxidative stress-related genes, such as the Txnip. The increased level of oxidative stress may have contributed to the histopathological and transcriptome changes of HPG axis organs in the prolonged neonatal hypothyroidism rats, especially in male pituitaries.

Peroxisome proliferator-activated receptor γ (PPARγ) is the master transcriptional regulator of adipocytes and the cellular target of thiazolidinedione (TZD) drugs. Suppression of pro-inflammatory actions, including pro-inflammatory gene expression and lipolysis in adipocytes, contributes to PPARγ-mediated anti-diabetic effects of TZDs. However, adverse side effects largely limited the clinical use of TZDs, despite their potent insulin-sensitizing effects. Therefore, it is important to understand how PPARγ is regulated. Thyroid hormone receptor-associated protein 3 (THRAP3) was previously reported to promote diabetic gene expression by acting as a transcriptional coregulator of PPARγ in adipocytes. Therefore, we tested if THRAP3 modulated anti-inflammatory functions of PPARγ in 3T3-L1 adipocytes. THRAP3 depletion increased basal and tumor necrosis factor α (TNFα)-induced lipolysis, pro-inflammatory gene expression, and phosphorylation of extracellular signal-regulated kinases (ERKs), suggesting elevated pro-inflammatory response after THRAP3 depletion in adipocytes. Moreover, TZD-mediated suppression of TNFα-induced lipolysis, pro-inflammatory gene expression, and ERK phosphorylation was attenuated or alleviated after THRAP3 depletion. Interestingly, the mRNA and protein levels of PPARγ were greatly reduced in THRAP3-depleted adipocytes. Actinomycin D treatment revealed that the stability of PPARγ mRNA was greatly reduced by THRAP3 depletion in adipocytes. Thus, in addition to modulating PPARγ function, THRAP3 may directly regulate the transcript of PPARγ in differentiated adipocytes.

Elevated thyroid-stimulating hormone (TSH) is associated with an increased risk of spontaneous abortion (SA); however, the associated mechanism remains unclear. This study aimed to investigate the expression of miRNAs and pathogenesis in the chorionic villi of TSH > 2.5 mIU/L-related SA patients. The chorionic villi were collected from pregnant women in the first trimester with TSH > 2.5 mIU/L with or without SA, as well as TSH < 2.5 mIU/L with or without SA to determine the level of miRNA expression. Differentially expressed miRNAs were confirmed by qRT-PCR in a total of 92 subjects. Cell Counting Kit-8 (CCK8), wound healing, transwell assays, and Western blotting were used to measure cellular biological functions and related proteins in HTR-8/SVneo cells. The potential mechanisms were determined using a Luciferase reporter assay and rescue experiment. Compared with normal pregnant women, miR-17-5p was decreased and zinc finger protein 367 (ZNF367) was upregulated in the chorionic villi of TSH > 2.5 mIU/L-related SA patients. Using HTR-8/SVneo cells, we demonstrated that elevated TSH inhibited miR-17-5p expression, as well as trophoblast migration and invasion. The overexpression of miR-17-5p targeted and inhibited ZNF367 expression promoting the biological function of trophoblasts. Further studies confirmed that ZNF367 interference partially reversed the biological function of the miR-17-5p inhibitor on HTR-8/SVneo cells. Taken together, our results showed that miR-17-5p promoted the biological function of trophoblasts by suppressing ZNF367.

Pentraxin 3 (PTX3) is a soluble pattern recognition receptor playing an important role in immune response and inflammation. Lipopolysaccharide (LPS) stimulation can significantly induce PTX3 expression and secretion in adipocytes. Appropriate regulation of PTX3 secretion is critical for inflammatory homeostasis. Using chemical inhibitors of conventional and unconventional protein secretion, we explored the mechanisms that control LPS-stimulated PTX3 secretion in 3T3-L1 adipocytes. Inhibiting the conventional protein secretion blocked LPS-stimulated PTX3 secretion, resulting in cellular PTX3 accumulation in adipocytes. We also detected PTX3 in exosomes from LPS-treated adipocytes; inhibiting exosome trafficking attenuated PTX3 secretion. However, only 4.3% of secreted PTX3 was detected in exosomes compared to 95.7% in the non-exosomal fractions. The fractionation of isolated exosomes by the iodixanol density gradient centrifugation confirmed that a small portion of secreted PTX3 overlapped with exosomal markers in small extracellular-vesicle fractions. We conclude that PTX3 is secreted mainly through conventional protein secretion, and a small percentage of PTX3 is released in exosomes from LPS-stimulated adipocytes.

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor that plays a fundamental role in extracellular calcium (Ca²⁺ _e) homeostasis by regulating parathyroid hormone release and urinary calcium excretion. The CaSR has been described to activate all four G protein subfamilies (Gα_q/11, Gα_i/o, Gα_12/13, Gα_s), and mutations in the receptor that cause hyper/hypocalcaemia, have been described to bias receptor signalling. However, many of these studies are based on measurements of second messengers or gene transcription that occurs many steps downstream of receptor activation and can represent convergence points of several signalling pathways. Therefore, to assess CaSR-mediated G protein activation directly, we took advantage of a recently described NanoBiT G protein dissociation assay system. Our studies, performed in HEK293 cells stably expressing CaSR, demonstrate that Ca²⁺ _e stimulation activates all Gα_q/11 family and several Gα_i/o family proteins, although Gα_z was not activated. CaSR stimulated dissociation of Gα_12/13 and Gα_s from Gβ-subunits, but this occurred at a slower rate than that of other Gα-subunits. Investigation of cDNA expression of G proteins in three tissues abundantly expressing CaSR, the parathyroids, kidneys and pancreas, showed Gα₁₁, Gα_z, Gα_i1 and Gα₁₃ genes were highly expressed in parathyroid tissue, indicating CaSR most likely activates Gα₁₁ and Gα_i1 in parathyroids. In kidney and pancreas, the majority of G proteins were similarly expressed, suggesting CaSR may activate multiple G proteins in these cells. Thus, these studies validate a single assay system that can be used to robustly assess CaSR variants and biased signalling and could be utilised in the development of new pharmacological compounds targeting CaSR.

Gouty arthritis is a common inflammatory disease characterized by monosodium urate (MSU) crystal-induced nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome activation with upregulated caspase 1 protease and IL-1β in macrophages. Cucurbitacin B (CuB) is a tetracyclic triterpene that possesses a potential anti-inflammatory activity. However, the immunomodulatory and anti-inflammatory effects of CuB on gout have not been well characterized. Therefore, the purpose of the present study was to determine whether CuB exhibits anti-inflammatory effects on gout and to analyze the underlying molecular mechanism. We examined the effects of CuB on various stimuli-activated bone marrow-derived macrophages (BMDMs) and in a mouse model with MSU-induced acute gouty arthritis. Our results demonstrated that CuB effectively suppressed multiple stimuli-activated IL-1β secretion by interrupting NLRP3 inflammasome complex formation, inhibiting NLRP3 inflammasome activation and suppressing key enzymes of glycolysis in macrophages. Consistent with this, CuB pretreatment also ameliorated MSU-induced arthritis in vivo models of gout arthritis, manifested by reduced foot swelling and inflammatory cell infiltration. Taken together, our data provide the evidence that CuB is an NLRP3 inflammasome inhibitor with therapeutic potential for treating NLRP3 inflammasome-mediated diseases, especially gouty arthritis.

In obesity, high levels of TNF-α in the bone marrow microenvironment induce the bone marrow-mesenchymal stem cells (BM-MSCs) towards a pro-adipogenic phenotype. Here, we investigated the effect of obesity on the migratory potential of BM-MSCs and their fate towards the adipose tissues. BM-MSCs were isolated from male C57Bl/06 mice with high-fat diet-induced obesity. The migratory potential of the BM-MSCs, their presence in the subcutaneous (SAT) and the visceral adipose tissues (VAT), and the possible mechanisms involved were investigated. Obesity did not affect MSC content in the bone marrow but increased the frequency of MSCs in blood, SAT, and VAT. In these animals, the SAT adipocytes presented a larger area, without any changes in adipokine production or the Sdf-1α gene expression. In contrast, in VAT, obesity increased leptin and IL-10 levels but did not modify the size of the adipocytes. The BM-MSCs from obese animals presented increased spontaneous migratory activity. Despite the augmented expression of Cxcr4, these cells exhibited decreased migratory response towards SDF-1α, compared to that of BM-MSCs from lean mice. The PI3K-AKT pathway activation seems to mediate the migration of BM-MSCs from lean mice, but not from obese mice. Additionally, we observed an increase in the spontaneous migration of BM-MSCs from lean mice when they were co-cultured with BM-HCs from obese animals, suggesting a paracrine effect. We concluded that obesity increased the migratory potential of the BM-MSCs and induced their accumulation in VAT, which may represent an adaptive mechanism in response to chronic nutrient overload.

Despite all modern advances in medicine, there are few reports of effective and safe drugs to treat obesity. Our objective was to screen anti-obesity natural compounds, and to verify whether they can reduce the body weight gain and investigate their molecular mechanisms. By using drug-screening methods, Phytohemagglutinin (PHA) was found to be the most anti-obesity candidate natural compound. Six-week-old C57BL/6J mice were fed with a high-fat diet (HFD) and intraperitoneally injected with 0.25 mg/kg PHA everyday for 8 weeks. The body weight, glucose homeostasis, oxygen consumption and physical activity were assessed. We also measured the heat intensity, body temperature and the gene expression of key regulators of energy expenditure. Prevention study results showed PHA treatment not only reduced the body weight gain but also maintained glucose homeostasis in HFD-fed mice. Further study indicated energy expenditure and uncoupling protein 1 (UCP-1) expression of brown adipose tissue (BAT) and white adipose tissue (WAT) in HFD-fed mice were significantly improved by PHA. In the therapeutic study, a similar effect was observed. PHA inhibited lipid droplet formation and upregulated mitochondrial-related gene expression during adipogenesis in vitro. UCP-1 KO mice displayed no differences in body weight, glucose homeostasis and core body temperature between PHA and control groups. Our results suggest that PHA prevent and treat obesity by increasing energy expenditure through upregulation of BAT thermogenesis.

Earlier, it was shown that reversing the downregulation of neuritin expression in the brain improves central neuropathy in diabetic rats. We investigated the protective mechanism of neuritin in diabetic cognitive dysfunction via astrocytes. Further, the impact of the overexpression of neuritin in the cortex and the hippocampus on diabetic cognitive dysfunction and astrogliosis in type 2 diabetic (db/db) mice was assessed. Antagonists were used to inhibit the JAK2/STAT3 signaling pathway in U-118MG, an astrocyte cell line. Immunofluorescence, Western blotting, and real-time PCR were performed. Neuritin overexpression in the hippocampus of db/db mice significantly ameliorated cognitive dysfunction, hippocampal neuronal impairment, and synaptic plasticity deterioration, and inhibited astrogliosis and the JAK2/STAT3 signaling pathway in the hippocampus. Neuritin suppressed the JAK2/STAT3 signaling pathway to inhibit lipopolysaccharide-induced gliosis in U-118MG cells. It was observed that neuritin regulates the JAK2/STAT3 signaling pathway in astrocytes to inhibit astrogliosis and improve diabetic cognitive dysfunction.

Glucocorticoid excess often causes a variety of cardiovascular complications, including hypertension, atherosclerosis, and cardiac hypertrophy. To abrogate its cardiac side effects, it is necessary to fully disclose the pathophysiological role of glucocorticoid in cardiac remodelling. Previous clinical and experimental studies have found that omentin-1, one of the adipokines, has beneficial effects in cardiovascular diseases, and is closely associated with metabolic disorders. However, there is no evidence to address the potential role of omentin-1 in glucocorticoid excess-induced cardiac injuries. To uncover the links, the present study utilized rat model with glucocorticoid-induced cardiac injuries and clinical patients with abnormal cardiac function. Chronic administration of glucocorticoid excess reduced rat serum omentin-1 concentration, which closely correlated with cardiac functional parameters. Intravenous administration of adeno-associated virus encoding omentin-1 upregulated the circulating omentin-1 level and attenuated glucocorticoid excess-induced cardiac hypertrophy and functional disorders. Overexpression of omentin-1 also improved cardiac mitochondrial function, including the reduction of lipid deposits, induction of mitochondrial biogenesis, and enhanced mitochondrial activities. Mechanistically, omentin-1 phosphorylated and activated the GSK3β pathway in the heart. From a study of 28 patients with Cushing’s syndrome and 23 healthy subjects, the plasma level of glucocorticoid was negatively correlated with omentin-1, and was positively associated with cardiac ejection fraction and fractional shortening. Collectively, the present study provided a novel role of omentin-1 in glucocorticoid excess-induced cardiac injuries and found that the omentin-1/GSK3β pathway was a potential therapeutic target in combating the side effects of glucocorticoid.