NAMPT enhances LOX expression and promotes metastasis in human chondrosarcoma cells by inhibiting miR-26b-5p synthesis.

Chondrosarcoma is a malignant bone tumor that emerges from abnormalities in cartilaginous tissue and is related with lung metastases. Nicotinamide phosphoribosyltransferase (NAMPT) is an adipocytokine reported to enhance tumor metastasis. Our results from clinical samples and the Gene Expression Omnibus data set reveal that NAMPT levels are markedly higher in chondrosarcoma patients than in normal individuals. NAMPT stimulation significantly increased lysyl oxidase (LOX) production in chondrosarcoma cells. Additionally, NAMPT increased LOX-dependent cell migration and invasion in chondrosarcoma by suppressing miR-26b-5p generation through the c-Src and Akt signaling pathways. Overexpression of NAMPT promoted chondrosarcoma metastasis to the lung in vivo. Furthermore, knockdown of LOX counteracted NAMPT-facilitated metastasis. Thus, the NAMPT/LOX axis presents a novel target for treating the metastasis of chondrosarcoma.

The effects of job characteristics on physicians' orientation toward lifelong learning.

With the rapidly growing body of medical knowledge, physicians must engage in lifelong learning. Physicians' orientation toward lifelong learning is of crucial importance. This study aimed to explore the effects of job characteristics on physicians' lifelong learning. A multicenter study collecting data from physicians from three medical centers in Taiwan was performed. A total of 321 physicians were surveyed with the Chinese version of the Job Content Questionnaire (C-JCQ) and the revised Jefferson Scale of Physician Lifelong Learning (JeffSPLL) to assess their job characteristics (i.e., job demands, job control, social support) and orientation toward lifelong learning. Exploratory factor analysis was employed to validate both questionnaires. Hierarchical regression was utilized to explore the relationship of job characteristics and predictors with physicians' lifelong learning. The results revealed that job demands (ß = 0.10), job control (ß = 0.19), social support from supervisors (ß = 0.16), the interaction of job demands × job control (ß = - 0.11) and the interaction of job demands × social support from colleagues (ß = 0.13) were significantly (p < .05, p < .001) related to lifelong learning. Moreover, physicians in the active group (high demand, high control) possessed a stronger orientation toward lifelong learning (mean = 3.57) than those in the low-strain group (mean = 3.42), high-strain group (mean = 3.39) and passive group (mean = 3.20). In conclusion, examining physicians' job demands, job control and social support helps us to understand their orientation toward lifelong learning and may provide insight to improve educational strategies.

A novel biomarker for pleural effusion diagnosis: Interleukin-36γ in pleural fluid.

BACKGROUND: Numerous studies have described the critical importance of interleukin (IL) -36γ in host defense against lung infections, but it is unknown whether it plays a role in infectious pleural effusion (IPE). This study aimed to examine the levels of IL-36γ in pleural effusions of different etiologies and evaluate the diagnostic accuracy of IL-36γ in the differential diagnosis of IPE. METHODS: A total of 112 individuals was enrolled in this research. IL-36γ levels in pleural fluids of all 112 patients were measured by enzyme-linked immunosorbent assay (ELISA). We also characterized these markers' diagnostic values across various groups. RESULTS: Patients with tuberculous pleural effusion (TPE) and parapneumonic effusion (PPE) had exhibited markedly higher IL-36γ levels in their pleural fluid than the malignant pleural effusion (MPE) and transudative effusion patients. Furthermore, the IL-36γ concentrations in TPE patients were evidently higher than in uncomplicated parapneumonic effusion (UPPE) patients but significantly lower than in complicated parapneumonic effusion (CPPE)/empyema patients. Pleural fluid IL-36γ is a useful marker to differentiate TPE from UPPE, at a cut-off value for 657.5 pg/ml (area under the curve = 0.904, p < 0.0001) with 70.0% sensitivity and 95.7% specificity. CONCLUSIONS: The elevated IL-36γ in pleural effusion may be used as a novel biomarker for infectious pleural effusion diagnosis, particularly in patients with CPPE/empyema, and is a potentially promising biomarker to differentiate between TPE and UPPE.

Follistatin (FST) is expressed in buffalo (Bubalus bubalis) ovarian follicles and promotes oocyte maturation and early embryonic development.

Follistatin (FST), a member of the transforming growth factor-ß (TGF-ß) superfamily, has been identified as an inhibitor of follicle-stimulating hormone. Previous studies showed that it plays an important role in animal reproduction. Therefore, this study aims to investigate its effect on the maturation of buffalo oocytes in vitro, and the underlying mechanism of FST affecting oocyte maturation was also explored in buffalo cumulus cells. Results showed that FST was enriched in the ovary and expressed at different stages of buffalo ovarian follicles as well as during oocyte maturation and early embryo development. The FST expression level was up-regulated in MII buffalo oocytes compared with the GV stage (p < .05). To study the effects of FST on buffalo oocytes' maturation and early embryonic development, we added the pcD3.1 skeleton vector and PCD3.1-EGFP-FST vector into the maturation fluid of buffalo oocytes, respectively. It was demonstrated that FST promoted the in vitro maturation rate of buffalo oocytes and the blastocyst rate of embryos cultured in vitro (p < .05). By interfering with FST expression, we discovered that FST in cumulus cells plays a crucial role in oocyte maturation. Interference with the FST expression during the buffalo oocyte maturation did not affect the first polar body rate of buffalo oocyte (p > .05). In contrast, the location of mitochondria in oocytes was abnormal, and the cumulus expansion area was reduced (p < .05). After parthenogenetic activation, the cleavage and blastocyst rates of the FST-interfered group were reduced (p < .05). Furthermore, RT-qPCR was performed to investigate further the underlying mechanism by which FST enhances oocyte maturation. We found that overexpression of FST could up-regulate the expression level of apoptosis suppressor gene Bcl-2 and TGF-ß/SMAD pathway-related genes TGF-ß, SMAD2, and SMAD3 (p < .05). In contrast, the expression levels of SMAD4 and pro-apoptotic gene BAX were significantly decreased (p < .05). The FST gene could affect buffalo oocyte maturation by regulating the oocyte mitochondria integrity, the cumulus expansion, cumulus cell apoptosis, and the expression levels of TGF-ß/SMAD pathway-related genes.

Identification of dominant conformational epitopes from the whole structure of the proprotein convertase subtilisin/kexin type 9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), a negative regulator of LDLR, has emerged as an important target for the treatment of hypercholesterolemic cardiovascular disease, and monoclonal antibodies alirocumab and evolocumab against it have been widely used in clinical practice. The vaccine research of PCSK9 is considered a promising option for the long-term treatment and prevention of cardiovascular disease, but progress has been slow. The selection of safe and effective epitopes is one of the key steps in vaccine development. In this study, we designed a phage display library of cascaded peptides for affinity screening with two antibody drugs, and found that the two peptides PC3 and PS6, which are adjacent to each other in protein spatial structure, both have superior binding activity to the screening antibodies. We performed in vitro recombination design on the dominant sequences, and obtained recombinant sequences that can respond to the dominant conformational epitope of PCSK9, which provides a meaningful reference for epitope selection in subsequent PCSK9 vaccine development.

Genes and compounds that increase type VII collagen expression as potential treatments for dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is a skin-blistering disease caused by mutations in COL7A1, which encodes type VII collagen (C7). There is no cure for DEB, but previous work has shown potential therapeutic benefit of increased production of even partially functional C7. Genome-wide screens using CRISPR-Cas9 have enabled the identification of genes involved in cancer development, drug resistance and other genetic diseases, suggesting that they could be used to identify drivers of C7 production. A keratinocyte C7 reporter cell line was created and used in a genome-wide CRISPR activation (CRISPRa) screen to identify genes and pathways that increase C7 expression. The CRISPRa screen results were used to develop a targeted drug screen to identify compounds that upregulate C7 expression. The C7_tdTomato cell line was validated as an effective reporter for detection of C7 upregulation. The CRISPRa screen identified DENND4B and TYROBP as top gene hits plus pathways related to calcium uptake and immune signalling in C7 regulation. The targeted drug screen identified several compounds that increase C7 expression in keratinocytes, of which kaempferol, a plant flavonoid, also significantly increased C7 mRNA and protein in DEB patient cells.

Small extracellular vesicles: from promoting pre-metastatic niche formation to therapeutic strategies in breast cancer.

Breast cancer is the most common cancer in females, and to date, the mortality rate of breast cancer metastasis cannot be ignored. The metastasis of breast cancer is a complex, staged process, and the pattern of metastatic spread is not random. The pre-metastatic niche, as an organ-specific home for metastasis, is a favourable environment for tumour cell colonization. As detection techniques improve, the role of the pre-metastatic niche in breast cancer metastasis is being uncovered. sEVs (small extracellular vesicles) can deliver cargo, which is vital for the formation of pre-metastatic niches. sEVs participate in multiple aspects of creating a distant microenvironment to promote tumour invasion, including the secretion of inflammatory molecules, immunosuppression, angiogenesis and enhancement of vascular permeability, as well as regulation of the stromal environment. Here, we discuss the multifaceted mechanisms through which breast cancer-derived sEVs contribute to pre-metastatic niches. In addition, sEVs as biomarkers and antimetastatic therapies are also discussed, particularly their use in transporting exosomal microRNAs. The study of sEVs may provide insight into immunotherapy and targeted therapies for breast cancer, and we also provide an overview of their potential role in antitumour metastasis. Video Abstract.

The eco-friendly electrosynthesis of trifluoromethylated spirocyclic indolines and their anticancer activity.

A method for the electrochemical diastereoselective oxytrifluoromethylation of indoles was developed for the eco-friendly synthesis of CF3-containing spirocyclic indolines. The cascade reaction comprised anodic oxidation to obtain CF3 radicals, the addition of radicals to indoles, and intramolecular spirocyclization. The reaction system without external chemical oxidants could easily be scaled up. Antiproliferation assays of these CF3-substituted spirocyclic indolines exhibited their promising activities and selectivities toward several types of cancer cells, including Huh-7, A549, and cisplatin-resistant cancer cells (A549/DDP).

MnSO4-promoted S-O bond cleavage for synthesizing functionalized sulfonium ylides from activated alkynes and sulfoxides.

A variety of functionalized sulfonium ylides were prepared in good yields through MnSO4-promoted S-O bond cleavage from activated alkynes and sulfoxides. Experimental results showed that the MnSO4 catalyst played important roles in accelerating the reaction and promoting the [1,3]-rearrangement of the S-O bond. Furthermore, the product was easily obtained on a gram scale by simple recrystallization without column chromatography. The obtained product can be converted to new sulfonium ylides and undergo cycloaddition with an alkyne to afford a trisubstituted furan scaffold.

Role of transforming growth factor-ß1 in recessive dystrophic epidermolysis bullosa squamous cell carcinoma.

Squamous cell carcinoma (SCC) develops in more than 80% of individuals with the skin blistering disorder recessive dystrophic epidermolysis bullosa (RDEB). In contrast with UV-induced SCC, RDEB-SCC results from skin damage and has a high proliferative and metastatic rate with 5-year survival near zero. Our objective is to determine the mechanisms underlying the increased metastatic tendencies of RDEB-SCC. RDEB-SCC cultured cell lines were treated with RDEB and non-RDEB fibroblast conditioned media and assayed for migration and invasion with and without small molecule inhibitors for TGFß and other downstream signal transduction pathways. TGFß1 secreted by RDEB dermal fibroblasts has been found to induce migration and invasion and to increase expression of epithelial-to-mesenchymal transition markers in an RDEB-SCC line. These effects were reversed upon inhibition of TGFß signalling and its downstream pathways MEK/ERK, P38 kinase and SMAD3. A number of small molecule inhibitors for these pathways are in different phases of various clinical trials and may be applicable to RDEB-SCC patients. Studying the mechanisms of the extreme form RDEB-SCC may inform studies of other types of SCC, as well as lead to better therapies for RDEB patients.

Neurotrophic keratitis in autoimmune polyglandular syndrome type 1: a case report.

BACKGROUND: Autoimmune polyglandular syndrome type 1 (APS-1) is a rare autosomal recessive disease. In patients with APS-1, the most frequently reported ocular manifestations are keratoconjunctivitis with dry eye and retinal degeneration. However, to our knowledge, no research studies have reported the relationship between APS-1 and neurotrophic keratitis (NK). Possible explanations such as limbus cell deficiency being the primary cause of APS-1 keratopathy are not applicable to our unusual case of the patient with APS-1 presenting as ocular surface disease with NK. Our case findings suggest a new explanation for the observed corneal pathology and a potential treatment for these patients. CASE PRESENTATION: A 27-year-old woman was referred to our hospital because of intermittent blurred vision and recalcitrant ocular surface problems in both eyes for many years. She has a history of autoimmune polyglandular syndrome type 1 (APS-1), which includes hypothyroidism, hypoparathyroidism, hypoadrenalism, and hypogonadotropic hypogonadism. In vivo confocal microscopy clearly demonstrated significant degeneration of the sub-basal nerve plexus and stromal nerve bundles in her corneas bilaterally. She was diagnosed with severe NK and ocular surface disease caused by dry eye. Treatment included the application of therapeutic soft contact lenses and punctual occlusion; however, both treatments had a limited effect. CONCLUSION: Patients with APS-1 may have ocular surface disease and severe damage to corneal nerves. Regular follow-up and treatment focusing on the regeneration of corneal nerves is particularly important in these patients.

Fine mapping of restorer-of-fertility gene based on high-density genetic mapping and collinearity analysis in pepper (Capsicum annuum L.).

KEY MESSAGE: The pepper restorer-of-fertility (CaRf) gene was fine mapped based on conjoint analysis of recombinants and collinearity between the two pepper reference genomes. Capana06g003028, encoding an Rf-like PPR protein, was proposed as the strongest candidate for pepper CaRf based on sequence comparison and expression analysis. The cytoplasmic male sterility (CMS)/restorer-of-fertility (Rf) system not only provides an excellent model to dissect genetic interactions between the mitochondria and nucleus but also plays a vital role in high-efficiency hybrid seed production in crops including pepper (Capsicum spp.). Although two important CMS candidate genes, orf507 and Ψatp6-2, have previously been suggested, the pepper Rf gene (CaRf) has not yet been isolated. In this study, a high-density genetic map comprising 7566 SNP markers in 1944 bins was first constructed with the array genotyping results from 317 F2 individuals. Based on this map, the CaRf gene was preliminarily mapped to a region of 1.15 Mb in length at the end of chromosome P6. Then, by means of a conjoint analysis of recombinants and collinearity between the two pepper reference genomes, an important candidate interval with 270.10 kb in length was delimited for CaRf. Finally, Capana06g003028, which encodes an Rf-like PPR protein, was proposed as the strongest candidate for CaRf based on sequence analysis and expression characteristics in sterile and fertile plants. The high-density genetic map and fine mapping results provided here lay a foundation for the application of molecular breeding, as well as cloning and functional analysis of CaRf, in pepper.

Theca cell-conditioned medium added to in vitro maturation enhances embryo developmental competence of buffalo (Bubalus bubalis) oocytes after parthenogenic activation.

Theca cells (TCs) play a key role in follicular growth and atresia. TCs synthesize androgens that act as substrate for granulosa cells (GCs) aromatization to estrogens needed for oocyte maturation. However, the effects of TCs in the form of conditioned medium on in vitro maturation (IVM) and developmental competence of buffalo oocytes remain unclear. In the present study, we examined the impacts of TC-conditioned medium (TCCM) on maturation efficiency and embryo development of buffalo oocytes after parthenogenic activation (PA). Our results showed that TCCM that was collected on day 2 and added to IVM medium at a 20% proportional level (2 days & 20%) exerted no significant effect on IVM rate (43.06% vs. 44.71%), but significantly (p  < .05) enhanced embryo development (oocyte cleavage, 80.93% vs. 69.66%; blastocyst formation, 39.85% vs. 32.84%) of buffalo oocytes after PA compared with the control group. However, monolayer TC significantly (p < .05) promoted both maturation efficiency (48.84% vs. 44.53%) and embryo development (oocyte cleavage, 80.39% vs. 69.32%; blastocyst formation, 35.38% vs. 29.25%) of buffalo oocytes after PA compared to that in the control group. Furthermore, TCs secreted some testosterone into the conditioned medium, which significantly (p < .05) promoted the expression levels of oestrogen synthesis-related genes (CYP11A1, CYP19A1 and 17ß-HSD) in buffalo cumulus-oocyte complexes (COCs). Our study indicated that TCCM (2 days & 20%) did not significantly affect IVM efficiency, but enhanced embryo developmental competence of oocytes after PA principally by stimulating the secretion of testosterone and facilitating estradiol synthesis of buffalo COCs.

A new three-dimensional glass scaffold increases the in vitro maturation efficiency of buffalo (Bubalus bubalis) oocyte via remodelling the extracellular matrix and cell connection of cumulus cells.

At present, many three-dimensional (3D) culture systems have been reported, improving the oocyte quality of in vitro maturation (IVM), yet the mechanism still needs to be further explored. Here we examined the effects of a new self-made 3D glass scaffold on buffalo oocyte maturation; meanwhile, the underlying mechanism on buffalo oocyte maturation was also detected. Compared to the two-dimensional (2D) glass dish culture, results revealed that the 3D culture can improve the first polar body rate of oocytes, subsequent cleavage and blastocysts rate of parthenogenetic activation embryos (p < .05). The extracellular matrix-related proteins COL1A1, COL2A1, COL3A1, FN and cell connection-related proteins N-cadherin, E-cadherin, GJA1 were found higher in cumulus cells of 3D culture. Moreover, in cumulus cells, proteins of the PI3K/AKT pathway reported being regulated by FN and E-cadherin including PI3K P85 and p-AKT were also higher in 3D culture. Furthermore, proapoptosis proteins P53, BAX, caspase-3 were lower in both cumulus cells and oocytes in 3D culture, while proteins PCNA and BCL2 showed the opposite result. Results also showed that the apoptosis was inhibited, and the proliferation was enhanced in cumulus cells of 3D culture. Finally, the cumulus expansion-related genes HAS2, CD44, HMMR, PTX3, PTGS2 were found higher in cumulus cells of 3D culture. Taken together, the 3D culture could promote oocyte maturation by regulating proteins correlated with the ECM, cell connection and PI3K/AKT pathway, inhibiting the apoptosis of cumulus cells and oocytes, enhancing the proliferation of cumulus cells and the cumulus expansion.

Clinical features and outcomes of Acanthamoeba keratitis in a tertiary hospital over 20- year period.

PURPOSE: To analyze the outcomes of Acanthamoeba keratitis (AK) in terms of different clinical presentations in a tertiary hospital in Taiwan over a 20- year period. METHODS: This is a retrospective case series. Patients with AK diagnosed at the National Taiwan University Hospital between January 1996 and December 2015 were identified. A diagnosis of AK was made on the basis of positive Acanthamoeba smear/cultures or pathological identification of Acanthamoeba cysts on keratoplasty specimens. Patient demographics, clinical presentations, treatment courses, and final visual outcomes were collected and analyzed. Visual acuity, postoperative complications and graft survivals were measured as outcomes. RESULTS: Of the 62 patients with AK in our study, 64.5% were medically treated while 35.5% underwent surgical treatment. In those with ring infiltrate, 52.4% patients could be successfully treated with medications. In eyes receiving penetrating keratoplasty, postoperative complications were more common in therapeutic penetrating keratoplasty (TPK) than those in optical penetrating keratoplasty (OPK) group (82.4% versus 40%, p = 0.04). The graft size was larger in TPK than that in OPK group (8.56 versus 7.83 mm, p = 0.002). Furthermore, post-operative glaucoma, which was the major complication, was found to be associated with larger graft size (p = 0.02) and dilated pupil/iris atrophy (p = 0.01). CONCLUSION: Even in advanced cases with ring infiltrate, eradication of infection with anti-amoebic drugs is possible. In those requiring keratoplasty, the surgical timing should be made meticulously considering graft size and signs of dilated pupil/iris atrophy in terms of post-operative glaucoma.

Therapeutic penetrating keratoplasty for microbial keratitis in Taiwan from 2001 to 2014.

PURPOSE: To study the surgical outcome of therapeutic penetrating keratoplasty (TPK) for medically uncontrolled microbial keratitis at a tertiary hospital in Taiwan over a 14-year period. METHODS: This is a retrospective case series study. Patients who underwent TPK at National Taiwan University Hospital in 2001-2014 were included. Patients were divided into 3 diagnostic groups: bacterial keratitis, fungal keratitis, and acanthamoeba keratitis. Each of the following criteria was evaluated: graft clarity at 1 month and 1 year postoperatively, cure of the disease, and anatomical success rate. RESULTS: A total of 107 TPKs were included. TPK eradicated the infection in 57/62 (91.9%) of bacterial keratitis, 33/41 (80.5%) of fungal keratitis, and 9/10 (90.0%) of acanthamoeba keratitis. 22/57 grafts (38.6%) of bacterial keratitis, 22/38 grafts (57.9%) of fungal keratitis, and 5/10 grafts (50.0%) of acanthamoeba keratitis remained clear at 1 year postoperatively. The 1-year graft survival rate did not significantly differ among these 3 groups. The leading causes of graft failure were late endothelial decompensation and graft reinfection. A higher percentage of graft clarity was achieved in grafts <8.5 mm in diameter than in larger grafts (55.4% vs. 31.4%, P = 0.027). Of the 14 patients with endophthalmitis at the time of TPK, the infection was successfully treated in 13 patients except 1 patient required evisceration. CONCLUSION: TPK is valuable in the management of medically uncontrolled microbial keratitis, although the 1-year graft survival rate is unsatisfactory. TPK combined with intravitreal antibiotics and vitrectomy is also a beneficial treatment strategy for patients with endophthalmitis secondary to microbial keratitis.

Dermatologic tacrolimus ointment on the eyelids for steroid-refractory vernal keratoconjunctivitis.

PURPOSE: The study aims to evaluate the therapeutic effects of dermatologic tacrolimus ointment on eyelids to treat refractory vernal keratoconjunctivitis (VKC). METHOD: This institutional study examined the effects of steroid treatment for 10 patients diagnosed with severe VKC refractory to steroid treatment. Patients received 0.1% dermatologic topical tacrolimus treatment on their eyelids once or twice daily for concomitant atopic dermatitis. The therapeutic outcomes were evaluated according to change in severity of clinical findings recorded with serial external ocular photography and change in requirement for steroid treatment. RESULTS: Clinical signs and symptoms improved substantially after tacrolimus treatment. Significant reduction in size of papillae, decrease of discharge, improvement in hyperemia, and shield ulcer healing with re-epithelization were observed in all patients. Six out of 10 (60%) patients did not receive steroid treatment. Long-term maintenance of tacrolimus was required to prevent episodic exacerbation. Patients' only treatment-related complaints were of mild burning sensations during medication application to eyelids, and this sensation disappeared a few days after treatment. CONCLUSIONS: Application of 0.1% dermatologic tacrolimus ointment to eyelids is effective and safe in the treatment of refractory VKC in patients with concomitant atopic dermatitis. This treatment may serve as a substitute for or decrease the requirement of steroid treatment.

Clinicopathologic correlation of ocular surface squamous neoplasia from a university hospital in North Taiwan 1994 to 2014.

BACKGROUND/PURPOSE: To describe the clinical and histologic characteristics of ocular surface squamous neoplasia (OSSN) and evaluate factors significant in recurrence at a university hospital in North Taiwan. METHODS: Patient charts, clinical features, and pathology records were retrospectively reviewed in patients with pathology-proved OSSN from January, 1994 to December, 2014. Clinicopathologic correlation was analyzed. RESULTS: Thirty-six patients were recruited. Mean age was 63.4 ± 13.0 (ranging from 23 to 87) years old. OSSN was predominant in men (21/36). Clinical appearances included papilliform in 17 eyes, gelatinous in 11 eyes, leukoplakic in 3 eyes, and 5 eyes in corneal intraepithelial neoplasia (CIN). Of 31 conjunctival OSSN, there were 4 in CIN I, 11 in CIN II, 13 in CIN III, and 3 in squamous cell carcinoma. Superior location was associated with higher-grade OSSN. Although statistical analysis was not significant, papilliform and multifocal lesions showed a trend of high-grade OSSN. The stages of tumor were 4, 5, 26, and 1 eye(s) in T1 to T4, respectively. Recurrence of disease occurred in 9 cases (25%) with mean recurrence time of 20.6 (range: 4 to 65) months. Multifocal lesion has a higher tendency for recurrence. CONCLUSION: Superior location was associated with high-grade OSSN, and papilliform OSSN might have a tendency of severe and invasive lesions. Multifocal lesions might be associated with higher-grade OSSN and higher recurrence rates.

Anti-Diabetic Effect of a Shihunine-Rich Extract of Dendrobium loddigesii on 3T3-L1 Cells and db/db Mice by Up-Regulating AMPK-GLUT4-PPARα.

The stems of Dendrobium loddigesii, a Chinese herb, are often used to treat diabetes and its polar extract is rich in shihunine, a water-soluble Orchidaceae alkaloid, but little is known about the anti-diabetes effects and mechanism of shihunine. This study investigated the anti-diabetic effect of a shihunine-rich extract of D. loddigesii (DLS) based on 3T3-L1 cells and db/db mice. The underlying mechanisms were primarily explored using Western blot analysis and immunohistochemical staining. The 3T3-L1 cell experiments showed that DLS can reduce the intracellular accumulation of oil droplets as well as triglycerides (p < 0.001) and promote the 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2deoxyglucose (2-NBDG) uptake of 3T3-L1 cells (p < 0.001). The animal experiments confirmed that after 8 weeks of DLS treatment, the body weight, fasting blood sugar, and serum lipid levels of mice were significantly lowered, and the oral glucose tolerance test and serum insulin level were significantly improved compared to the no-treatment diabetes mellitus group. Further histomorphology observation led to the conclusion that the quantities of islet cells were significantly increased and the increase in adipose cell size was significantly suppressed. The immunohistochemical test of pancreatic tissue revealed that DLS inhibited the expression of cleaved cysteine aspartic acid-specific protease 3 (cleaved caspase-3). Western blot experiments showed that DLS had agonistic effects on adenosine monophosphate (AMP)-activated protein kinase phosphorylation (p-AMPK) and increased the expression levels of peroxisome proliferator-activated receptor α (PPARα) and glucose transporter 4 (GLUT4) in liver or adipose tissues. These data suggest that the shihunine-rich extract of D. loddigesii is an anti-diabetic fraction of D. loddigesii. Under our experimental condition, DLS at a dose of 50 mg/kg has good anti-diabetic efficacy.