Self-assembly of differentiated dental pulp stem cells facilitates spheroid human dental organoid formation and prevascularization.

BACKGROUND: The self-assembly of solid organs from stem cells has the potential to greatly expand the applicability of regenerative medicine. Stem cells can self-organise into microsized organ units, partially modelling tissue function and regeneration. Dental pulp organoids have been used to recapitulate the processes of tooth development and related diseases. However, the lack of vasculature limits the utility of dental pulp organoids. AIM: To improve survival and aid in recovery after stem cell transplantation, we demonstrated the three-dimensional (3D) self-assembly of adult stem cell-human dental pulp stem cells (hDPSCs) and endothelial cells (ECs) into a novel type of spheroid-shaped dental pulp organoid in vitro under hypoxia and conditioned medium (CM). METHODS: During culture, primary hDPSCs were induced to differentiate into ECs by exposing them to a hypoxic environment and CM. The hypoxic pretreated hDPSCs were then mixed with ECs at specific ratios and conditioned in a 3D environment to produce prevascularized dental pulp organoids. The biological characteristics of the organoids were analysed, and the regulatory pathways associated with angiogenesis were studied. RESULTS: The combination of these two agents resulted in prevascularized human dental pulp organoids (Vorganoids) that more closely resembled dental pulp tissue in terms of morphology and function. Single-cell RNA sequencing of dental pulp tissue and RNA sequencing of Vorganoids were integrated to analyse key regulatory pathways associated with angiogenesis. The biomarkers forkhead box protein O1 and fibroblast growth factor 2 were identified to be involved in the regulation of Vorganoids. CONCLUSION: In this innovative study, we effectively established an in vitro model of Vorganoids and used it to elucidate new mechanisms of angiogenesis during regeneration, facilitating the development of clinical treatment strategies.

Influence of macrophage polarization in herniated nucleus pulposus tissue on clinical efficacy after lumbar discectomy.

Background: Low back pain or sciatic pain because of lumbar intervertebral disc herniation (LDH) is caused by mechanical compression and/or an inflammatory component on the nerve root. However, it is difficult to define to what extent each component contributes to the pain. This study attempted to explore the effects of macrophage polarization on clinical symptoms in patients experiencing LDH after surgery, and investigated the association between macrophage cell percentages and clinical efficacy. Methods: This study retrospectively harvested nucleus pulposus (NP) tissue samples from 117 patients. Clinical symptoms and efficacy using the visual analog scale (VAS) and Oswestry Disability Index (ODI) were evaluated at different time points preoperatively and postoperatively. CD68, CCR7, CD163, and CD206 were selected as macrophage phenotypic markers. Results: Seventy-six samples showed positive expression of macrophage markers in NP samples of patients with LDH, whereas 41 patients displayed negative results. No significant differences were detected between the two groups, involvement of several demographic data, and preoperative clinical findings. With respect to the macrophage-positive group, no significant correlation was detected between the positive rate of the four markers and the VAS score or ODI after surgery. However, patients with NP samples positive for CD68 and CCR7 expression showed significantly lower VAS scores 1 week after surgery compared with those in the negative group. Moreover, the improvement in VAS score showed a strong positive correlation with CD68- and CCR7-positive cell percentages. Conclusions: Our results indicated that pro-inflammatory M1 macrophages may be associated with the reduction of chronic pain after surgery. Therefore, these findings contribute to better personalized pharmacological interventions for patients with LDH, considering the heterogeneity of pain.

Investigation of macrophage polarization in herniated nucleus pulposus of patients with lumbar intervertebral disc herniation.

Macrophage infiltration and polarization during lumbar intervertebral disc herniation (LDH) have attracted increased attention but their role remains unclear. To explore macrophage polarization in herniated nucleus pulposus (NP) tissue of patients with LDH and investigate the association between cell frequency and different clinical characteristics or symptoms, we conducted a retrospective study by analyzing NP tissue samples from 79 patients. Clinical features and symptoms, using the visual analog scale (VAS) and Oswestry disability index (ODI), were collected. The macrophage markers CD68, CCR7, CD163, and CD206; pro-inflammatory cytokine TNF-α; and anti-inflammatory factor IL-4 were analyzed by immunohistochemistry. The frequency of polarized macrophages and positivity rate of pro- and anti-inflammatory cytokines showed significant differences in some of clinical characteristics. Specifically, higher CCR7+ and TNF-α + proportions were identified in the high-intensity zone (HIZ) and the type of extrusion and sequestration NP tissue than in non-HIZ and protrude NP tissue. Higher CD206+ and IL-4+ proportion were detected in Modic changes. However, no differences in gender, age, smoking status, Pfirrmann grade, analgesic use, leg pain duration, and segments were found between groups. CD68+ , CCR7+ , and CD206+ cell proportions, and TNF-α and IL-4 showed positive associations with VAS scores preoperation. Associations between ODI and the macrophages markers were weak/insignificant. Our results indicated that macrophage polarization or macrophage-like cells contribute to LDH pathological features. Macrophage populations displaying significant associations with VAS score reflected continuous M1/M2 transition contributing to pain during LDH. These findings may contribute to enhanced/personalized pharmacological interventions for patients with LDH considering pain heterogeneity.

Bindarit Reduces Bone Loss in Ovariectomized Mice by Inhibiting CCL2 and CCL7 Expression via the NF-κB Signaling Pathway.

OBJECTIVE: To investigate the changes in proinflammatory cytokines and chemokines, namely, C-C motif ligand (CCL) 2 and CCL7, in postmenopausal osteoporosis (PMOP) and to develop a new drug, bindarit (Bnd), for PMOP in an ovariectomized (OVX) mouse model. METHODS: Bone marrow macrophages (BMMs) from the femurs of five women with PMOP and five premenopausal women without osteoporosis were detected by RNA sequencing. BMMs from mice were differentiated into osteoclasts and treated with a synthetic inhibitor of CCL2 and CCL7, Bnd, or 17 beta estradiol (E2 ). Mouse BMMs were differentiated into osteoclasts with or without Bnd for 7 days and analyzed by RNA sequencing. Osteoblasts of mice were induced to undergo osteoblastogenesis and treated with Bnd. OVX mice were treated with E2 or Bnd after surgery. The protein and mRNA expression of CCL2 and CCL7 was detected using immunostaining and qPCR, respectively, in OVX and aged mice and in cells cultured in vitro. Osteoclast formation was detected using a tartrate-resistant acid phosphatase (TRAP) assay in vitro and in vivo. Alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) were detected using immunostaining to evaluate osteogenesis. Microcomputed tomography was conducted to analyze trabecular bone parameters, the structure model index, bone mineral density and other variables. Nuclear factor-κB (NF-κB) signaling pathway-related protein phosphorylation of IKKα/ß (p-IKKα/ß) and p-NFκB p65 was examined using western blotting. RESULTS: CCL2, CCL7 and their receptor of C-C chemokine receptor-2 (CCR2), and the NF-κB signaling pathway, were significantly increased in women with PMOP. CCL2 and CCL7 protein and mRNA expression was increased in OVX mice and aged female mice, but the increases were attenuated by E2 and Bnd. E2 and Bnd effectively inhibited osteoclastogenesis and the protein expression of CCL2 and CCL7 both in vitro and in vivo and reduced bone loss in OVX mice. Bnd did not affect the mineralization of osteoblasts directly in vitro but reduced bone turnover in vivo. p-IKKα/ß and p-NFκB p65 levels were increased in BMMs of mice after differentiation into osteoclasts but were significantly decreased by Bnd. CONCLUSION: The proinflammatory cytokines and chemokines CCL2, CCL7 and CCR2 were correlated with PMOP. Bnd attenuated the increases in CCL2 and CCL7 levels to affect osteoporosis in OVX mice via the NFκB signaling pathway. Thus, Bnd may be useful as a new therapeutic for the prevention of PMOP.

Acute EPA-induced learning and memory impairment in mice is prevented by DHA.

Eicosapentaenoic acid (EPA), an omega-3 fatty acid, has been widely used to prevent cardiovascular disease (CVD) and treat brain diseases alone or in combination with docosahexaenoic acid (DHA). However, the impact of EPA and DHA supplementation on normal cognitive function and the molecular targets of EPA and DHA are still unknown. We show that acute administration of EPA impairs learning and memory and hippocampal LTP in adult and prepubescent mice. Similar deficits are duplicated by endogenously elevating EPA in the hippocampus in the transgenic fat-1 mouse. Furthermore, the damaging effects of EPA are mediated through enhancing GABAergic transmission via the 5-HT6R. Interestingly, DHA can prevent EPA-induced impairments at a ratio of EPA to DHA similar to that in marine fish oil via the 5-HT2CR. We conclude that EPA exhibits an unexpected detrimental impact on cognitive functions, suggesting that caution must be exercised in omega-3 fatty acid supplementation and the combination of EPA and DHA at a natural ratio is critical for learning and memory and synaptic plasticity.

N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide promotes bone formation via the canonical Wnt/ß-catenin signaling pathway.

Osteoporosis is characterized by low bone mineral density and microarchitectural deterioration of bone tissue. N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide (MBOC) is one of the macamides isolated from Maca (Lepidium meyenii Walp.), a cruciferous plant from the Andes of Peru. In this study, C3H/10T1/2 mesenchymal stem cells were treated with MBOC in osteogenic induction medium. An ovariectomized (OVX) mouse model was used to investigate the effect of 1-month MBOC treatment on the prevention of postmenopausal osteoporosis. Remarkably, trabecular thickness, trabecular number, and bone volume/tissue volume of the distal femoral metaphysis were significantly increased in OVX + MBOC mice compared with OVX mice, as revealed by microcomputed tomography analysis. Trabecular separation was decreased in OVX + MBOC mice compared with OVX mice. Consistently, MBOC increased the levels of osteocalcin and runt-related transcription factor 2 in OVX mice, as well as the expression of runt-related transcription factor 2, osterix, and alkaline phosphatase in C3H/10T1/2 cells. Mechanistically, MBOC activates the canonical Wnt/ß-catenin signaling pathway via inhibiting phosphorylation of GSK-3ß at Tyr216 and maintaining ß-catenin expression. Collectively, the current study demonstrates the robustness of MBOC in the induction of mesenchymal stem cells osteogenic differentiation and consequent bone formation, suggesting that MBOC may be a potentially effective drug to treat postmenopausal osteoporosis.

B-cell-specific mammalian target of rapamycin complex 1 activation results in severe osteoarthritis in mice.

This study aims to investigate the effect of enriched plasma cells on the production of inflammatory cytokines and development of osteoarthritis (OA) in mice with B-cell-specific conditional deletion of the tuberous sclerosis 1 gene (TSC1). OA was induced by destabilization of the medial meniscus (DMM) in mice with TSC1 disruption in B cells (CD19-TSC1) and in littermate control mice (CON). The effects of DMM and incidence of OA were evaluated histologically, mRNA levels of inflammatory cytokines were detected by polymerase chain reaction, and serum cytokine levels were detected by enzyme-linked immunosorbent assay. Deletion of TSC1 caused constitutive activation of mechanistic target of rapamycin complex 1 mTORC1 in B cells. CON mice subjected to DMM exhibited a severe OA phenotype with increased inflammatory cytokines in B cells, serum, and the synovial membrane. Importantly, inflammatory cytokine production was also increased in B cells from the spleen of CD19-TSC1 conditional KO mice, but the OA phenotype was significantly elevated in conditional KO mice after DMM surgery compared with CON mice, as indicated by more severe articular cartilage destruction, increased protein expression of matrix metalloproteinase-13 and mRNA of type X collagen in the articular cartilage, decreased mRNA expression of type II collagen in the articular cartilage, and increased inflammatory cytokines in serum and the synovial membrane. The results demonstrate that inflammatory cytokine synthesis by B cells was enriched in CD19-TSC1 conditional KO mice, and this enhanced synthesis of inflammatory cytokines accelerated the incidence of OA.

[Estradiol significantly increases the expression of antioxidant enzymes in osteoporotic rats and osteoblasts in vitro].

OBJECTIVE: To investigate the effect of estradiol on the expression of antioxidant enzymes in osteoblasts and its role in postmenopausal osteoporosis. METHODS: Rat models of osteoporosis established by ovariectomy were treated with estradiol for 3 months, and the changes in serum levels of reactive oxygen species (H2O2) and antioxidant enzymes (γ -GCS, GSH-ST and GSH-px) were detected. The effects of estradiol on the expression of γ -GCS mRNA and protein in osteoblast-like cells MC3T3-E1, MG63 and OB were examined with PCR and Western blotting. Using a mRNA microarray, we analyzed the changes in the expressions of 84 antioxidant enzymes in the osteoblast cell line MC3T3-E1 following estradiol treatment, and the enzymes with significant changes were verified by PCR. CCK-8 kit was used to evaluate the effect of estradiol and antioxidant NAC on the proliferation of MC3T3-E1 cells. RESULTS: Rat models of osteoporosis were successfully established with ovariectomy. The osteoporotic rats showed significantly increased serum level of reactive oxygen species (H2O2) and decreased levels of antioxidant enzymes. Estrogen treatment of the osteoporotic rats obviously reversed the phenotype of osteoporosis, lowered serum level of reactive oxygen species, and increased the level of γ -GCS. In MC3T3-E1, MG63 and OB cells, estradiol treatment significantly upregulated the expression levels of γ -GCS mRNA and protein. In MC3T3-E1 cells treated with estrogen, the mRNA chip identified 6 upregulated antioxidant enzymes (Gpx6, Gstk1, Nos2, Prdx2, Ngb and Ccs), and the results of PCR verified that estradiol upregulated Ccs and Ngb mRNAs in MC3T3-E1, MG63 and OB cells. Estradiol and antioxidant NAC obviously promoted the proliferation of MC3T3-E1 cells. CONCLUSION: Estradiol significantly increases the expression of antioxidase γ -Gcs, Ccs and Ngb in osteoblasts in vitro. Postmenopausal osteoporosis is closely related with the increase of reactive oxygen species and the decrease of antioxidant levels. In osteoblasts, estrogen deficiency may increase the level of reactive oxygen species, decrease the level of antioxidant enzymes, activate the oxidative stress cascade, and consequently inhibit the proliferation of osteoblasts to aggravate the condition of osteoporosis.

[A method for efficient transduction of miR-483-5p in the kidney of mice].

OBJECTIVE: To establish a method for gene delivery in murine renal tissue using lentivirus vector encoding miR-483-5p. METHODS: Thirty-five C57BL/6J mice were randomly divided into control group, low-dose treatment group (5 µL each kidney) , and high?dose treatment group (20 µL each kidney), and in the latter two groups, the lentivirus vector encoding miR-483-5p were injected in the renal cortex. The tissue samples were collected at 7 and 21 days after the injection. A transgenic mouse model with inducible systemic overexpression of miR-483-5p was established in TG483 mice. The Cre-loxp system was used to create a mouse model with renal tubule-specific expression of miR-483-5p. The levels of BUN in the mice were detected and HE staining and fluorometric TUNEL assay were used to observe the morphological changes of the kidneys; real-time qPCR was used to detect miR-483-5p expression in the renal cortex. RESULTS: The mice with overexpression of miR-483-5p had normal renal function without obvious pathological changes or apoptosis in the renal tissue. Renal cortex injection of 20 µL lentivirus resulted in obviously increased level of miR-483-5p at 21 days (1.2∓0.43 vs 8.6∓1.09, P<0.001). miR-483-5p showed a low expression (0.9∓0.09 vs 1.7∓0.19, P<0.05) in TG483 mice and a high expression in the kidney of the transgenic mice established using the Cre-loxp system (1.6∓1.13 vs 12.36∓3.89, P<0.05). CONCLUSION: The transgenic mice with renal tubule-specific expression of miR-483-5p show normal renal function, and this model facilitates further study of the role of miR-483-5p in the kidney.

[Rictor/mTORC2 regulates blood-testis barrier and spermatogenesis in mice].

OBJECTIVE: To investigate the role of Rictor/mTORC2 in the formation of blood testis barrier (BTB), testicular development, and spermatogenesis. METHODS: Amh Cre positive mice homozygous for rictor loxP with Sertoli cell specific deletion of rictor were obtained by cross breeding Amh Cre mice with rictor loxP mice. The histology of the reproductive organs, seminiferous tubules and epididymis of the transgenic mice was observed with HE staining. The cell subgroups of the germ cells in the seminiferous tubule were detected by flow cytometry with propidium iodide labeling. The expression levels of Ki 67 and separase were detected with immunofluorescence assay, and the expression levels of BTB associated proteins were detected with immunofluorescence and Western blotting. RESULTS: Compared with the control (Amh Cre-, rictorloxP/loxP or rictorloxP/-) mice, the mice with Sertoli cell specific rictor deletion showed significantly decreased testicular weight and epididymis weight (P<0.05), significantly increased diploid cells (P<0.01), and decreased haploid cells (P<0.01) but comparable tetraploid cells and similar expression levels of Ki 67 and separase. The mice with rictor knockout also showed aberrant localization of BTB associated proteins, which were scattered over the whole seminiferous epithelium, but the expression levels of the protein remained stable. CONCLUSION: Rictor in testicular Sertoli cells is essential for maintaining BTB integrity and function and ensuring normal spermatogenesis in mice.

Identification of novel genetic loci for osteoporosis and/or rheumatoid arthritis using cFDR approach.

There are co-morbidity between osteoporosis (OP) and rheumatoid arthritis (RA). Some genetic risk factors have been identified for these two phenotypes respectively in previous research; however, they accounted for only a small portion of the underlying total genetic variances. Here, we sought to identify additional common genetic loci associated with OP and/or RA. The conditional false discovery rate (cFDR) approach allows detection of additional genetic factors (those respective ones as well as common pleiotropic ones) for the two associated phenotypes. We collected and analyzed summary statistics provided by large, multi-center GWAS studies of FNK (femoral neck) BMD (a major risk factor for osteoporosis) (n = 53,236) and RA (n = 80,799). The conditional quantile-quantile (Q-Q) plots can assess the enrichment of SNPs related to FNK BMD and RA, respectively. Furthermore, we identified shared loci between FNK BMD and RA using conjunction cFDR (ccFDR). We found strong enrichment of p-values in FNK BMD when conditional Q-Q was done on RA and vice versa. We identified 30 novel OP-RA associated pleiotropic loci that have not been reported in previous OP or RA GWAS, 18 of which located in the MHC (major histocompatibility complex) region previously reported to play an important role in immune system and bone health. We identified some specific novel polygenic factors for OP and RA respectively, and identified 30 novel OP-RA associated pleiotropic loci. These discovery findings may offer novel pathobiological insights, and suggest new targets and pathways for drug development in OP and RA patients.

Rheb1 deletion in myeloid cells aggravates OVA-induced allergic inflammation in mice.

The small GTPase ras homolog enriched in brain (Rheb) is a downstream target of tuberous sclerosis complex 1/2 (TSC1/2) and an upstream activator of the mechanistic target of rapamycin complex 1 (mTORC1), the emerging essential modulator of M1/M2 balance in macrophages. However, the role and regulatory mechanisms of Rheb in macrophage polarization and allergic asthma are not known. In the present study, we utilized a mouse model with myeloid cell-specific deletion of the Rheb1 gene and an ovalbumin (OVA)-induced allergic asthma model to investigate the role of Rheb1 in allergic asthma and macrophage polarization. Increased activity of Rheb1 and mTORC1 was observed in myeloid cells of C57BL/6 mice with OVA-induced asthma. In an OVA-induced asthma model, Rheb1-KO mice demonstrated a more serious inflammatory response, more mucus production, enhanced airway hyper-responsiveness, and greater eosinophil numbers in bronchoalveolar lavage fluid (BALF). They also showed increased numbers of bone marrow macrophages and BALF myeloid cells, elevated M2 polarization and reduced M1 polarization of macrophages. Thus, we have established that Rheb1 is critical for the polarization of macrophages and inhibition of allergic asthma. Deletion of Rheb1 enhances M2 polarization but decreases M1 polarization in alveolar macrophages, leading to the aggravation of OVA-induced allergic asthma.

Neuronal mTORC1 Is Required for Maintaining the Nonreactive State of Astrocytes.

Astrocytes respond to CNS insults through reactive astrogliosis, but the underlying mechanisms are unclear. In this study, we show that inactivation of mechanistic target of rapamycin complex (mTORC1) signaling in postnatal neurons induces reactive astrogliosis in mice. Ablation of Raptor (an mTORC1-specific component) in postmitotic neurons abolished mTORC1 activity and produced neurons with smaller soma and fewer dendrites, resulting in microcephaly and aberrant behavior in adult mice. Interestingly, extensive astrogliosis without significant astrocyte proliferation and glial scar formation was observed in these mice. The inhibition of neuronal mTORC1 may activate astrogliosis by reducing neuron-derived fibroblast growth factor 2 (FGF-2), which might trigger FGF receptor signaling in astrocytes to maintain their nonreactive state, and FGF-2 injection successfully prevented astrogliosis in Raptor knock-out mice. This study demonstrates that neuronal mTORC1 inhibits reactive astrogliosis and plays an important role in CNS pathologies.

[Docosahexaenoic acid inhibits aflatoxin B1-induced migration and invasion in hepatocellular carcinoma cells in vitro].

OBJECTIVE: To investigate the effect of docosahexaenoic acid (DHA) on invasiveness of aflatoxin B1 (AFB1)-induced hepatocellular carcinoma cells in vitro. METHODS: HepG2.2.15 cells were exposed to different concentrations of AFB1 and DHA plus AFB1. The cell migration and invasion were assessed using wound-healing and Transwell assay, and flow cytometry was used to analyze the cell cycle changes. The ultrastructural changes of the cells were observed by transmission electron microscopy. RESULTS: Compared with the control group, the cells exposed to2 µmol/L AFB1 showed obviously enhanced migration and invasion with decreased cell ratio in G1/G1 phase and increased cell ratio in G2/M phase but no changes in S phase cells; transmission electron microscopy revealed the presence of multiple nucleoli and significantly increased mitochondria and Golgi apparatus in the exposed cells. Compared with AFB1-exposed cells, the cells treated with DHA and AFB1 showed decreased migration and invasion abilities, and the G1/G1 phase cells increased and G2/M phase cells decreased significantly; ultrastructurally, the cells contained single nucleoli with decreased mitochondria and vacuolization occurred in the cytoplasm. CONCLUSION: DHA can significantly inhibit AFB1-induced enhancement of cell migration and invasion in hepatocellular carcinoma cells in vitro.

[Chemical Constituents from Drypetes hainanensis Stems and Leaves].

OBJECTIVE: To study the chemical constituents from the stems and leaves in Drypetes hainanensis. METHODS: The constituents were isolated and purified by various chromatography, and the structures were identified by extensive spectral analysis. RESULTS: Eleven compounds were isolated and identified as syringaresinol-4-O-glycoside (1), koaburaside (2), abietin (3) syringin (4), kelampayoside A (5), 7,7'-bis-(4-hydroxy-3,5-dimethoxyphenyl)-8,8'-dihydroxymethyl-tetrahydrofuran-4-O-ß-glucopyranoside (6), amentoflavone (7), 3,4,5-trimethoxyphenyl-ß-D-glucopyranoside (8),1,4-di-O-methyl-myo-inositol (9), glycerol (10) and succinic acid (11). CONCLUSION: All the compounds are isolated from this plant for the first time.

DEPTOR expression negatively correlates with mTORC1 activity and tumor progression in colorectal cancer.

The mammalian target of rapamycin (mTOR) signaling pathway is upregulated in the pathogenesis of many cancers, including colorectal cancer (CRC). DEPTOR is an mTOR inhibitor whose expression is negatively regulated by mTOR. However, the role of DEPTOR in the development of CRC is not known. The aim of this study was to investigate the expression of DEPTOR and mTORC1 activity (P-S6) in a subset of CRC patients and determine their relation to tumor differentiation, invasion, nodal metastasis and disease-free survival. Here, Immunohistochemical expression of P-S6 (S235/236) and DEPTOR were evaluated in 1.5 mm tumor cores from 90 CRC patients and in 90 samples of adjacent normal mucosa by tissue microarray. The expression of P-S6 (S235/236) was upregulated in CRC, with the positive rate of P-S6 (S235/236) in CRC (63.3%) significantly higher than that in control tissues (36.7%, 30%) (p<0.05). P-S6 (S235/236) also correlated with high tumor histologic grade (p=0.002), and positive nodal metastasis (p=0.002). In contrast, the expression level of DEPTOR was correlated with low tumor histological grade (p=0.006), and negative nodal metastasis (p=0.001). Interestingly, P-S6 (S235/236) expression showed a significant negative association with the expression of DEPTOR in CRC (p=0.011, R= -0.279). However, upregulation of P-S6 (S235/236) (p=0.693) and downregulation of DEPTOR (p=0.331) in CRC were not significantly associated with overall survival. Thus, we conclude that expression of DEPTOR negatively correlates with mTORC1 activity and tumor progression in CRC. DEPTOR is a potential marker for prognostic evaluation and a target for the treatment of CRC.

Endogenous n-3 polyunsaturated fatty acids protect against imiquimod-induced psoriasis-like inflammation via the IL-17/IL-23 axis.

The beneficial effects of n-3 polyunsaturated fatty acids (PUFAs) on psoriasis have been reported in rats, mice and humans, but the specific mechanisms involved have not been well defined. The present study utilized the fat-1 mouse, a transgenic model that can endogenously convert n-6 FAs into n-3 PUFAs, to directly determine if the outcomes of psoriasis were correlated with n-3 PUFAs. Wild-type (WT) and fat-1 mice, which were treated daily with imiquimod (IMQ) cream or control cream on the shaved right ear and dorsal skin, were fed the same diet. The severity of inflammation of the ear and dorsal skin was scored according to the clinical Psoriasis Area and Severity Index (PASI) and epidermal hyperplasia was measured by H&E staining. The expression of inflammatory factors in the epidermis was analyzed by immunohistochemical analysis. Flow cytometry and an enzyme-linked immunosorbent assay were used to measure the differences in the content of inflammatory factors in the blood serum and to determine which of CD4+ T cells were present in the spleen between IMQ-induced fat-1 mice and WT mice. Fat-1 IMQ-induced mice exhibited significantly lower levels of inflammatory cell-like T helper 17 cells (Th17 cells) and higher levels of regulatory T cells (Treg cells) in the spleen as compared with the WT IMQ-induced mice. n-3 fatty acids stimulated Th17 cells to produce lower levels of inflammatory factors, including interleukin (IL)-17, IL-22, IL-23 and stimulated Treg cells to produce higher anti-inflammatory factors, such as Foxp3. In conclusion, the present study provides further insight into the mechanisms involved in preventing inflammation in psoriasis-like mice by n-3 PUFAs using a fat-1 transgenic mouse model.

Enhancement of the synthesis of n-3 PUFAs in fat-1 transgenic mice inhibits mTORC1 signalling and delays surgically induced osteoarthritis in comparison with wild-type mice.

BACKGROUND: An exogenous supplement of n-3 polyunsaturated fatty acids (PUFAs) has been reported to prevent osteoarthritis (OA) through undefined mechanisms. OBJECTIVE: This study investigated the effect of alterations in the composition of endogenous PUFAs on OA, and associations of PUFAs with mammalian target of rapamycin complex 1 (mTORC1) signalling, a critical autophagy pathway in fat-1 transgenic (TG) mice. METHODS: fat-1 TG and wild-type mice were used to create an OA model by resecting the medial meniscus. The composition of the endogenous PUFAs in mouse tissues was analysed by gas chromatography, and the incidence of OA was evaluated by micro-computed tomography (micro-CT), scanning electron microscopy and histological methods. Additionally, primary chondrocytes were isolated and cultured. The effect of exogenous and endogenous PUFAs on mTORC1 activity and autophagy in chondrocytes was assessed. RESULTS: The composition of endogenous PUFAs of TG mice was optimised both by increased n-3 PUFAs and decreased n-6 PUFAs, which significantly alleviated the articular cartilage destruction and osteophytosis in the OA model (p<0.01), decreased protein expression of matrix metalloproteinase-13 (MMP-13) and ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs) in the articular cartilage (p<0.01) and reduced chondrocyte number and loss of cartilage extracellular matrix. Both exogenous and endogenous n-3 PUFAs downregulated mTORC1 activity and promoted autophagy in articular chondrocytes. Conversely, mTORC1 pathway activation suppressed autophagy in articular chondrocytes. CONCLUSIONS: Enhancement of the synthesis of endogenous n-3 PUFAs from n-6 PUFAs can delay the incidence of OA, probably through inhibition of mTORC1, promotion of autophagy and cell survival in cartilage chondrocytes. Future investigation into the role of the endogenous n-6/n-3 PUFAs composition in OA prevention and treatment is warranted.

Endogenous n-3 polyunsaturated fatty acids (PUFAs) mitigate ovariectomy-induced bone loss by attenuating bone marrow adipogenesis in FAT1 transgenic mice.

AIM: To investigate the effect of endogenous n-3 polyunsaturated fatty acids (PUFAs) on bone marrow adipogenesis under osteoporosis conditions. METHODS: A mouse osteoporosis model overexpressing the FAT1 gene from Caenorhabditis elegans and converting n-6 PUFAs to n-3 PUFAs endogenously was used. RESULTS: The mice presented significantly lower bone marrow adiposity (adipocyte volume/tissue volume, mean adipocyte number) but increased the bone parameters (bone mineral density, bone mineral content, bone volume/total volume) in the distal femoral metaphysis. CONCLUSION: Endogenous n-3 PUFAs protect bone marrow adipogenesis, which provides a novel drug target.

Establishing a disc degeneration model using computed tomography-guided percutaneous puncture technique in the rabbit.

BACKGROUND: Various animal models have been developed to investigate the complex mechanisms leading to intervertebral disc disorders and to evaluate the different therapeutic options. The needle puncture technique is commonly used to induce intervertebral degeneration in animal models. The present study aimed to establish a rabbit model of intervertebral disc degeneration using a simple, minimally invasive procedure. METHODS AND MATERIALS: The animal model was created in the rabbit using computed tomography-guided percutaneous puncture technology. An 18-gauge needle was used to induce a disc injury with a 5-mm puncture depth. Radiographic, histologic, and biochemical analyses and magnetic resonance imaging were performed to assess the consequent disc degeneration. RESULTS: Significant disc space narrowing was observed as early as 4 wk, and osteophytes were formed at 12 wk after puncture. The magnetic resonance imaging assessment demonstrated a progressive loss of T2-weighted signal intensity at the stabbed discs throughout the 12-wk period. The histologic analysis showed a progressive loss of the normal architecture from 4 wk to the end point. The biochemical assays suggested that the expression of proteoglycan decreased progressively with increasing time. CONCLUSIONS: A simple, but minimally invasive, intervertebral disc degeneration model was established successfully using computed tomography-guided percutaneous puncture technology in the rabbit. The puncture procedure can be performed with minimal damage and handling of the other structures, ensuring a uniform reproducible disc degeneration model.