Tardiphaga alba sp. nov., a heavy-metal-tolerant bacterium isolated from garden soil.

A previously undescribed, heavy-metal-tolerant, motile, Gram-negative bacterium, designated strain SK50-23T, was characterized using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SK50-23T was closely related to Tardiphaga robiniae LMG 26467T and the non-phototrophic 'Rhodopseudomonas boonkerdii' NS23T (98.1 and 97.3 % 16S rRNA gene sequence similarity, respectively). Strain SK50-23T possessed a circular genome of 5.86 Mb, with a DNA G+C content of 61.9 mol%. Digital DNA-DNA hybridization showed 20.8-21.6 % similarity between strain SK50-23T and related species. In addition, the whole-genome average nucleotide identity values between strain SK50-23T and related species ranged from 75.1 to 83.5 %. The major cellular fatty acid identified in strain SK50-23T was C18 : 1ω7c, and the main isoprenoid quinone present was ubiquinone Q-10. Strain SK50-23T could be assigned to the genus Tardiphaga with the species name Tardiphaga alba sp. nov. based on morphological, chemotaxonomic and genome-based taxonomic characteristics, and 16S rRNA gene-based phylogenetic characteristics. The type strain of the proposed novel species is SK50-23T (=NBRC 108825T=CGMCC No. 1.12037T).

Volatile oil of Angelica sinensis Radix improves cognitive function by inhibiting miR-301a-3p targeting Ppp2ca in cerebral ischemia mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica Sinensis Radix (ASR) is a commonly used Chinese medicine known for its effects on tonifying blood, promoting blood circulation, and alleviating pain associated with menstrual regulation. Additionally, it has been used in the treatment of vascular cognitive impairment (VCI). The primary pharmacodynamic agent within ASR is volatile oil of Angelica Sinensis Radix (VOASR), which has demonstrated efficacy in combating cognitive impairment, although its mechanism remains unclear. OBJECTIVE: This study aimed to elucidate the potential molecular mechanisms underlying VOASR's improvement of cognitive function in cerebral ischemic mice. METHODS: A model of cerebral ischemic mice was established through unilateral common carotid artery occlusion (UCCAO) surgery, followed by intervention with VOASR. Cognitive function was assessed using the Morris water maze (MWM) test, while RT-qPCR was utilized to measure the differential expression of miR-301a-3p in the hippocampus. To evaluate cognitive function and hippocampal protein differences, wild-type mice and miR-301a-3p knockout mice were subjected to the MWM test and iTRAQ protein profiling. The relationship between miR-301a-3p and potential target genes was validated through a Dual-Luciferase Reporter experiment. RT-qPCR and Western blot were employed to determine the differential expression of Ppp2ca and synaptic plasticity-related proteins in the mouse hippocampus. RESULTS: Intervention with VOASR significantly improved cognitive impairment in cerebral ischemic mice and reduced the expression of miR-301a-3p in the hippocampus. Our findings suggest that miR-301a-3p may regulate cognitive function by targeting Ppp2ca. Furthermore, VOASR intervention led to an increase in the expression of Ppp2ca and synaptic plasticity-related proteins. CONCLUSION: Our study indicates that VOASR may be involved in regulating cognitive function by inhibiting miR-301a-3p, consequently increasing the expression of Ppp2ca and synaptic plasticity proteins. These results provide a new target and direction for the treatment of cognitive dysfunction.

Novel-miR-81 Promotes the Chondrocytes Differentiation of Bone Marrow Mesenchymal Stem Cells Through Inhibiting Rac2 Expression.

OBJECTIVE: MicroRNAs (miRNAs) play a key role in the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into chondrocytes. Our previous study found that novel-miR-81 can relieve osteoarthritis, but its role in chondrogenic differentiation of BMSCs remains unclear. The purpose of this study was to explore the role of novel-miR-81 in chondrogenic differentiation of BMSCs. METHODS: We used a model in which transforming growth factor (TGF)-ß3-induced BMSCs differentiation into chondrocytes. We detected the expression Sox9, Collagen Ⅱ, Aggrecan, novel-miR-81, and Rac2 by real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Western blot was performed to detect the expression of Sox9, Collagen Ⅱ, and Rac2. Dual-luciferase reporter gene assay confirmed that the association between novel-miR-81 and Rac2. In addition, the ectopic chondrocyte differentiation of BMSCs was performed subcutaneously in nude mice. The effect of novel-miR-81 and Rac2 on ectopic chondrogenic differentiation of BMSCs was determined by immunohistochemical staining. RESULTS: Novel-miR-81 upregulated in chondrogenic differentiation of BMSCs. Rac2 was a key target of novel-miR-81. Mimic novel-miR-81 and siRac2 upregulated the expression of Sox9, Collagen Ⅱ, and Aggrecan. CONCLUSION: Novel-miR-81 promotes the chondrocytes differentiation of BMSCs by inhibiting the expression of target gene Rac2, which provides potential targets for BMSCs transplantation to repair cartilage defects.

Cervical lymph node metastasis of bladder cancer: a case report and review of literature.

OBJECTIVE: To report an extremely rare case of bladder cancer patient with cervical lymph nodes, abdominal lymph nodes, and bone metastases at the same time. METHODS AND RESULTS: The case was investigated by follow-up and immunohistochemistry was used in the pathological part. RESULT: The patient was diagnosed with bladder cancer (high-grade urothelial metastatic epithelial cell carcinoma) by pathology and immunohistochemistry after transurethral resection of bladder tumor (TURBT) and metastatic bladder cancer by pathology and immunohistochemistry after cervical lymph node aspiration due to neck lymph node enlargement 1 year later, and a CT of the chest and abdomen suggested that the patient also had abdominal lymph node and bone metastases.At the 2.5-year regular chemotherapy follow-up, the patient showed that the abdominal lymph node metastasis disappeared, the cervical lymph node fusion shrank, and the bone metastasis still existed. CONCLUSION: 1. Regular postoperative review is particularly important; 2.For patients with UCB who undergo TURBT, a effective regular perfusion program should be performed throughout the postoperative period; 3. For patients with postoperative metastatic symptoms of UCB, Complex treatment has a positive effect on patient prognosis; 4.The presence of enlarged head and neck lymph nodes in patients with bladder cancer should also be considered as metastatic of UCB.

Neuroprotective effect of aloe emodin against Huntington's disease-like symptoms in R6/1 transgenic mice.

Aloe emodin is a natural anthraquinone derived from aloe or rhubarb, showing anti-renal fibrosis, anti-atherosclerosis and anti-cancer effects. Aloe emodin also shows neuroprotective effects in ischemic stroke rats. Naturally, anthraquinone derivatives generally have the effect of inhibiting the transforming growth factor-ß1 (TGF-ß1) pathway. There is an increase in the calcium/calmodulin-dependent protein kinase II (CaMKII) and TGF-ß1 levels in both Huntington's disease (HD) patients' brains and HD transgenic mice. Thus, we hypothesized that aloe emodin may inhibit the phosphorylation of CaMKII (p-CaMKII) and TGF-ß1/sma- and mad-related protein (Smad) signaling in the brain, further preventing motor and cognitive dysfunction. Aloe emodin was orally administered to 10- to 20-week-old HD R6/1 transgenic mice. Aloe emodin improved the motor coordination of R6/1 transgenic mice in the rotarod test and attenuated visual recognition impairment in the novel object recognition test. Aloe emodin downregulated levels of the mutant huntingtin protein, p-CaMKII and TGF-ß1, but not the TGF-ß2 or TGF-ß3 levels, in the brains of R6/1 mice. Aloe emodin could also inhibit neuronal apoptosis in the hippocampus of R6/1 mice. Altogether, these results indicated that aloe emodin prevents several HD-like symptoms through the inhibition of CaMKII/Smad and TGF-ß1/Smad signaling in mice.

Paenibacillus sedimenti sp. nov., isolated from freshwater wetland sediment.

A Gram-stain-positive, motile, rod-shaped, facultatively anaerobic bacterium, designated strain WST5T, isolated from sediment was characterized using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain WST5T was most closely related to Paenibacillus aestuarii CJ25T (96.8 % similarity). The genome size of the WST5T was 6.5 Mb, contained 4500 predicted protein-coding genes, and had a DNA G+C content of 46.6%. The values of whole-genome average nucleotide identity analysis and digital DNA-DNA hybridization between strain WST5T and its closely related type strains were less than 76 and 25.6 %, respectively. The predominant cellular fatty acids (>10 %) were anteiso-C15 : 0 and C16 : 1 ω5c and the main menaquinone was MK-7. The major polar lipids were identified as diphospholidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and two unknown aminophospholipids. Based on the results of phenotypic, genotypic, chemotaxonomic and phylogenetic analyses, strain WST5T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus sedimentum sp. nov. is proposed. The type strain is WST5T (=NBRC 115194 T=CGMCC 1.18706T).

Bioinformatics analysis reveals lipid metabolism may play an important role in the SiO2-stimulated rat model.

Silicosis is a progressive and irreversible common occupational disease caused by long-term inhalation of a large amount of free silica dust. Its pathogenesis is complex, and the existing prevention and treatment methods can not effectively improve silicosis injury. To uncover potential differential genes in silicosis, SiO2-stimulated rats and their control original transcriptomic data sets GSE49144, GSE32147 and GSE30178 were downloaded for further bioinformatics analysis. We used R packages to extract and standardize transcriptome profiles, then screened differential genes, and enriched GO and KEGG pathways through clusterProfiler packages. In addition, we investigated the role of lipid metabolism in the progression of silicosis by qRT-PCR validation and transfection with si-CD36. A total of 426 differential genes were identified in this study. Based on GO and KEGG enrichment analysis, it was found that lipid and atherosclerosis were significantly enriched. qRT-PCR was used to detect the relative expression level of differential genes in this signaling pathway of silicosis rat models. mRNA levels of Abcg1, Il1b, Sod2, Cyba, Cd14, Cxcl2, Ccl3, Cxcl1, Ccl2 and CD36 increased, mRNA levels of Ccl5, Cybb and Il18 decreased. In addition, at the cellular level, SiO2-stimulated lead to lipid metabolism disorder in NR8383, and silencing CD36 inhibited SiO2-induced lipid metabolism disorder. These results indicate that lipid metabolism plays an important role in the progression of silicosis, and the genes and pathways reported in this study may provide new ideas for the pathogenesis of silicosis.

Homologous cancer cell membrane-camouflaged nanoparticles target drug delivery and enhance the chemotherapy efficacy of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common digestive tract malignancy that seriously threatens human life and health. Early HCC may be treated by intervention, surgery, and internal radiotherapy, while the choice for late HCC is primarily chemotherapy to prolong patient survival. Lenvatinib (LT) is a Food and Drug Administration (FDA)-approved frontline drug for the treatment of advanced liver cancer and has achieved excellent clinical efficacy. However, its poor solubility and severe side effects cannot be ignored. In this study, a bionic nanodrug delivery platform was successfully constructed. The platform consists of a core of Lenvatinib wrapped with a pH-sensitive polymer, namely, poly(ß-amino ester)-polyethylene glycol-amine (PAE-PEG-NH2), and a shell formed by a cancer cell membrane (CCM). The prepared nanodrugs have high drug loading capacity, long-term stability, good biocompatibility, and a long retention time. In addition, the targeting effect of tumor cell membranes and the pH-responsive characteristics of the polymer materials enable them to precisely target tumor cells and achieve responsive release in the tumor microenvironment, which makes them suitable for effective drug delivery. In vivo experiments revealed that the nanodrug showed superior tumor accumulation and therapeutic effects in subcutaneous tumor mice model and could effectively eliminate tumors within 21 days. As a result, it opens up a new way to reduce side effects and improve the specific therapeutic effect of first-line clinical medications to treat tumors.

rno-miR-90 promotes chondrogenic differentiation of bone marrow mesenchymal stem cells by targeting SPARC-related modular calcium binding 2.

Bone marrow mesenchymal stem cells (BMSCs) have the ability to differentiate into chondrocytes. In the differentiation of BMSCs into chondrocytes, micro-RNAs (miRNAs) play an important role. rno-miR-90 is a new miRNA discovered by our research team, and its role in chondrogenic differentiation of BMSCs is unknown. This study aimed to investigate whether rno-miR-90 could promote chondrogenic differentiation of BMSCs by regulating secreted protein acidic and rich in cysteine-related modular calcium binding 2 (Smoc2). First, BMSCs chondroblast differentiation was successfully induced in vitro by classical induction method of transforming growth factor (TGF)-ß3. On this basis, we transfected rno-miR-90 mimic and inhibitor, and confirmed that rno-miR-90 mimic could promote the differentiation of BMSCs into chondrocytes by real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. In addition, we demonstrated that Smoc2 was a target gene of rno-miR-90 by dual-luciferase reporter assay, and confirmed that rno-miR-90 mimic could inhibit the expression of Smoc2 by RT-qPCR and western blotting. In order to further prove the targeting relationship between rno-miR-90 and Smoc2, we constructed three interfering fragments of Smoc2, and proved that silencing Smoc2 could promote the differentiation of BMSCs into chondrocytes at the transcriptional and protein levels. Finally, we constructed a carrier scaffold for ectopic chondrogenic differentiation in vivo, and confirmed that rno-miR-90 mimic and siSmoc2 could promote chondrogenic differentiation of BMSCs by Alcian blue staining and immunohistochemistry. In summary, our results suggested that rno-miR-90 could promote chondrogenic differentiation of BMSCs by down-regulating the expression of Smoc2. rno-miR-90 mimic and Smoc2 may be therapeutic targets of osteoarthritis.

A facile method for anti-cancer drug encapsulation into polymersomes with a core-satellite structure.

Polymersomes possess the self-assembly vesicular structure similar to liposomes. Although a variety of comparisons between polymersomes and liposomes in the aspects of physical properties, preparation and applications have been elaborated in many studies, few focus on their differences in drug encapsulation, delivery and release in vitro and in vivo. In the present work, we have provided a modified direct hydration method to encapsulate anti-cancer drug paclitaxel (PTX) into PEG-b-PCL constituted polymersomes (PTX@PS). In addition to advantages including narrow particle size distribution, high colloid stability and moderate drug-loading efficiency, we find that the loaded drug aggregate in small clusters and reside through the polymersome membrane, representing a unique core-satellite structure which might facilitate the sustained drug release. Compared with commercial liposomal PTX formulation (Lipusu®), PTX@PS exhibited superb tumor cell killing ability underlain by multiple pro-apoptotic mechanisms. Moreover, endocytic process of PTX@PS significantly inhibits drug transporter P-gp expression which could be largely activated by free drug diffusion. In glioma mice models, it has also confirmed that PTX@PS remarkably eradicate tumors, which renders polymersomes as a promising alternative to liposomes as drug carriers in clinic.

Silibinin Ameliorates Formaldehyde-Induced Cognitive Impairment by Inhibiting Oxidative Stress.

Silibinin is a flavonoid extracted from the medicinal plant Silybum marianum (milk thistle), traditionally used to treat liver disease. Recent studies have shown that the antioxidative stress and anti-inflammatory effects of milk thistle are used in the treatment of neurological diseases. Silibinin has antioxidative stress and antiapoptotic effects and reduces cognitive impairment in models of Alzheimer's disease (AD). However, the underlying mechanism of silibinin related to improvement of cognition remains poorly understood. In this study, we used the model of lateral ventricle injection of formaldehyde to examine the related mechanism of silibinin in improving cognitive impairment disorders. Oral administration of silibinin for three weeks significantly attenuated the cognitive deficits of formaldehyde-induced mice in a Y-maze test and Morris water maze test. Y-maze results show that silibinin increases the rate of spontaneous response alternation in FA-induced mice. Silibinin increases the target quadrant spending time and decreases escape latency in the Morris water maze test. We examined the effect of silibinin on the NRF2 signaling pathway, and silibinin promoted the nuclear transfer of NRF2 and increased the expression of HO-1 but did not significantly increase the protein expression of NRF2 in the hippocampus. Well, silibinin reduces the content of DHE and decreases the levels of apoptosis of mature neuron cells. We investigated the effect of silibinin on the content of formaldehyde degrading enzymes; biochemical analyses revealed that silibinin increased GSH and ALDH2 in formaldehyde-induced mice. In addition, as one of the pathological changes of AD, TAU protein is also hyperphosphorylated in FA model mice. Silibinin inhibits the expression of GSK-3ß in model mice, thereby reducing the phosphorylation of TAU proteins ser396 and ser404 mediated by GSK3ß. Based on our findings, we verified that the mechanism of silibinin improving cognitive impairment may be antioxidative stress, and silibinin is one of the potentially promising drugs to prevent formaldehyde-induced cognitive impairment.

Curcumin protects thymus against D-galactose-induced senescence in mice.

Senescence-related decline of thymus affects immune function in the elderly population and contributes to the prevalence of many relevant diseases like cancer, autoimmune diseases, and other chronic diseases. In this study, we investigated the therapeutic effects of curcumin, an agent that could counter aging, and explored its optimal intake and the alteration of autoimmune regulator (Aire) after curcumin treatment in the D-galactose (D-gal)-induced accelerated aging mice. ICR mice were intraperitoneally injected with D-gal for 8 weeks to establish the accelerated aging model and given curcumin with 50, 100, and 200 mg/kg body weight per day by gavage, respectively, for 6 weeks. It indicated that the D-gal-treated mice developed structural changes in the thymi compared with the control group without D-gal and curcumin treatment. As the supplements of curcumin, it resulted in a restoration of the normal thymic anatomy with an increase of proliferating cells and a reduction of apoptotic cells in the thymi of the D-gal-induced aging model mice. Curcumin administration could also expand the expression level of Aire from mRNA level and protein level. The current study demonstrated that curcumin could ameliorate senescence-related thymus involution via upregulating Aire expression, suggesting that curcumin can rejuvenate senescence-associated alterations of thymus induced by D-gal accumulation.

Gallic acid attenuates thymic involution in the d-galactose induced accelerated aging mice.

Senescence is an inevitable and complicated phenomenon. Age-associated thymic involution increases the risk of infectious diseases, which results in the immunosenescence and leads to a poor immune function. d-galactose (d-gal) can cause damages that resemble accelerated aging in mice. Gallic acid (GA), as one of the natural phenolic compounds, has been demonstrated to act in antioxidant and anti-tumor effects. In this study, we explored the effects of GA in preventing the age-related thymic involution and the alterations of the forkhead box protein N1 (FoxN1) in d-gal induced accelerated aging mice. The accelerated aging mice model was established by intraperitoneal injection d-gal for eight weeks and given GA with 200, 250, 500 mg/kg body weight per day, respectively, for six weeks. It showed that the d-gal-treated mice developed structural changes in the thymi compared to normal control mice. With supplement of GA, the mice restored the normal thymic anatomy, including the thickening cortex compartment and clearer cortico-medullary junction. The d-gal-treated mice showed a severe reduction in the number of thymocytes, GA mice also displayed the increased numbers of CD4 + T cells through flow cytometric analysis. GA treatment increased the proliferative cells by BrdU incorporation assay and reduced the numbers of apoptotic cells with FITC-12-dUTP labeling (TUNEL). The expression of FoxN1 was also found increased in GA treated mice by immunohistochemistry and quantitative reverse transcriptase PCR (qRT-PCR). Taken together, our results suggested that the administration of GA opposed the involution of thymus via stimulation of FoxN1 expression and proliferation of cells in a dose-dependent manner.

Effects of reaction conditions on light-dependent silver nanoparticle biosynthesis mediated by cell extract of green alga Neochloris oleoabundans.

Silver nanoparticles (AgNPs) were synthesized by incubating the mixture of AgNO3 solution and whole-cell aqueous extracts (WCAEs) of Neochloris oleoabundans under light conditions. By conducting single-factor and multi-factor optimization, the effects of parameters including AgNO3 concentration, pH, and extraction time were quantitatively evaluated. The optimal conditions in terms of AgNP yield were found to be 0.8 mM AgNO3, pH 5, and 9-h extraction. The AgNPs thus synthesized were quasi-spherical with a mean particle diameter of 16.63 nm and exhibited decent uniformity as well as antibacterial activities, which may facilitate AgNP biosynthesis's application in the near future.

The expression of VE-cadherin in breast cancer cells modulates cell dynamics as a function of tumor differentiation and promotes tumor-endothelial cell interactions.

The cadherin switch has profound consequences on cancer invasion and metastasis. The endothelial-specific vascular endothelial cadherin (VE-cadherin) has been demonstrated in diverse cancer types including breast cancer and is supposed to modulate tumor progression and metastasis, but underlying mechanisms need to be better understood. First, we evaluated VE-cadherin expression by tissue microarray in 392 cases of breast cancer tumors and found a diverse expression and distribution of VE-cadherin. Experimental expression of fluorescence-tagged VE-cadherin (VE-EGFP) in undifferentiated, fibroblastoid and E-cadherin-negative MDA-231 (MDA-VE-EGFP) as well as in differentiated E-cadherin-positive MCF-7 human breast cancer cell lines (MCF-VE-EGFP), respectively, displayed differentiation-dependent functional differences. VE-EGFP expression reversed the fibroblastoid MDA-231 cells to an epithelial-like phenotype accompanied by increased ß-catenin expression, actin and vimentin remodeling, increased cell spreading and barrier function and a reduced migration ability due to formation of VE-cadherin-mediated cell junctions. The effects were largely absent in both MDA-VE-EGFP and in control MCF-EGFP cell lines. However, MCF-7 cells displayed a VE-cadherin-independent planar cell polarity and directed cell migration that both developed in MDA-231 only after VE-EGFP expression. Furthermore, VE-cadherin expression had no effect on tumor cell proliferation in monocultures while co-culturing with endothelial cells enhanced tumor cell proliferation due to integration of the tumor cells into monolayer where they form VE-cadherin-mediated cell contacts with the endothelium. We propose an interactive VE-cadherin-based crosstalk that might activate proliferation-promoting signals. Together, our study shows a VE-cadherin-mediated cell dynamics and an endothelial-dependent proliferation in a differentiation-dependent manner.

Polarized actin and VE-cadherin dynamics regulate junctional remodelling and cell migration during sprouting angiogenesis.

VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis.

[Active ingredients of Plastrum Testudinis inhibit epidermal stem cell apoptosis in serum-deprived culture].

OBJECTIVE: To investigate the effects of active ingredients of Plastrum Testudinis (PT) on serum deprivation-induced apoptosis of epidermal stem cells (ESCs). METHODS: ESCs were isolated from the back skin of fetal Sprague-Dawley rats with 2 weeks of gestational age and were divided into normal group (10% fetal bovine serum), control group (serum-deprived culture) and groups treated with serum deprivation plus active ingredients of PT, including ethyl acetate extract (2B), stearic acid ethyl ester (S6), tetradecanoic acid sterol ester (S8) and (+)-4-cholesten-3-one (S9). The vitality of ESCs after 24, 48 and 72 h of culture was measured with MTT method; apoptotic ESCs double-stained with Annexin V-FITC and propidium iodine were detected by flow cytometry (FCM); Bcl-2 and caspase-3 expressions were measured by Western blotting. RESULTS: MTT results indicated that the vitality of ESCs in the active ingredients of PT groups at 48 h was increased compared with the control group and 2B had better effects than the others. FCM results indicated that 2B had the most significant anti-apoptotic effect compared with the control as well as S6, S8 and S9. Western blot results indicated that 2B, S6, S8 and S9 up-regulated the expression of Bcl-2 protein and down-regulated the expression of caspase-3 protein compared with the control. CONCLUSION: Ethyl acetate extract of Plastrum Testudinis inhibits epidermal stem cell apoptosis in serum-deprived culture by regulating the expressions of Bcl-2 and caspase-3 proteins and has a stronger anti-apoptotic effect than its constituents S6, S8 and S9.

[Effects and mechanism of Plastrum testudinis extracts on PC12 apoptosis].

OBJECTIVE: To observe the inhibitive effects of Plastrum testudinis Extracts (PTE) on 6-Hydroxydopamine (6-OHDA) induced PC12 cells apoptosis and explore its mechanism. METHODS: PC12 apoptosis model was established by serum starvation and damaged for 24 hours. The cells were randomly divided into four groups:control group, 6-OHDA group, PTE 3, 30 microg/mL group. Cell optical density was determined by MTT; Ratio of cell apoptosis was examined by Annexin V/PI double stain flow cytometry (FCM), and Western blot was applied to detect the BCL-X/L expression. RESULTS: MTT and FCM analysis demonstrated that PTE can elevate PC12 cells viability and reduce their apoptotic ratio in a dose dependent manner. Western blot showed that PTE promoted the expression of BCL-X/L. CONCLUSION: PTE can inhibit the apoptosis of PC12 induced by 6-OHDA in a dose dependent manner, and its mechanism maybe associated partially with up-regulating BCL-X/L signaling pathway.

BMP-Id pathway targeted by cholesterol myristate suppresses the apoptosis of PC12 cells.

Identifying small molecules that suppress apoptosis is promising for the therapy of brain diseases. We recently showed that autocrine bone morphogenetic protein (BMP) signaling involves the effects of cholesterol myristate present in traditional Chinese medicine on mesenchymal stem cells. The present study evaluated the effects of cholesterol myristate on the apoptosis and BMP signaling of PC12 cells. PC12 cells transfected by the inhibitor of differentiation (Id1) promoter reporter construct target gene of BMP4 signaling; cholesterol myristate increases the activity of Id1 promoter. However, structurally related steroids such as cholesterol, ß-sitosterol and cholesten-3-one, lack of the myristate, did not affect the activity of Id1 promoter, suggesting that myristate is essential for the effect of cholesterol myristate. These effects depend on BMP signaling. Apoptosis analysis indicated that cholesterol myristate inhibited the apoptosis of PC12 cells induced in serum-free condition. Cholesterol myristate significantly increases the expression of BMP4, BMPRIA, p-Smad1/5/8, Id1 and its antiapoptotic target gene Bcl-xL in PC12 cells treated in serum-free condition. Moreover, BMP antagonist reduced the anti-apoptotic effect of cholesterol myristate. Thus, this study is to provide evidence that BMP-Id pathway targeted by cholesterol myristate suppresses the apoptosis of PC12 cells. Our findings are therefore of considerable therapeutic significance and provide the potential of newly exploiting cholesterol myristate and clinically in brain disease therapies.

Apigenin accelerates lipopolysaccharide induced apoptosis in mesenchymal stem cells through suppressing vitamin D receptor expression.

BACKGROUND: Transplantation of mensenchymal stem cells (MSCs) has been proposed as a promising way for tissue engineering. However, the application of MSCs for transplantation will undergo apoptosis due to the extremely harsh microenvironment such as excessive inflammation. Apigenin (API) has been reported to protect cells against inflammatory damage and cell death by exhibiting anti-inflammatory and anti-oxidative capacity. Here we investigated the modulatory effects of API in lipopolysaccharide (LPS)-mediated inflammation and apoptosis of MSCs, and further defined the underlying mechanism. METHODS: Effects of different concentrations of API (0, 5, 10, 20, 40 and 80 µmol/L) for 24 hours, and LPS (0, 0.5 and 5.0 µg/ml) for 6 hours and 24 hours on MSCs viability were assayed by MTT. Based on this, MSCs were pretreated with different concentrations of API (0 - 40 µmol/L) at the indicated times (6, 12 and 24 hours) followed by exposure to 5 µg/ml LPS for 24 hours. MTT, phase-contrast microscopy, annexinV/propidium iodide (PI) double stain flow cytometry (FCM) and Hoechst staining were applied to explore the effects of API on MSCs induced by 5 µg/ml LPS for 24 hours. In addition, reverse-transcription polymerase chain reaction (RT-PCR) was applied to detect the mRNA expression of pro-inflammatory factors including cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), nuclear factor-kappa B (NF-κB), pro-apoptotic gene caspase-3, Bad, and anti-apoptotic gene Bcl-2. Moreover, AutoDock software was used to imitate the docking score of API and vitamin D receptor (VDR). In parallel, Western blotting and RT-PCR were used to investigate protein and mRNA expression of VDR. RESULTS: MSCs stimulated with LPS 5 µg/ml for 24 hours was used as a model of apoptosis induced by over inflammatory stimulus. API (0 - 40 µmol/L) had non-toxic effect on MSCs; however, it could decrease mRNA expression of COX-2, iNOS and NF-κB at different time points in MSCs induced by LPS, except for API at the concentration of 5 µmol/L. RESULTS: from phase-contrast microscopy, MTT, Hoechst staining and AnnexinV/PI double stain FCM demonstrated that with the increasing concentrations of API and extension of administrating time, significant morphological changes of MSCs occurred, viability of cells was strongly inhibited, and meanwhile, apoptosis of LPS-administrated MSCs was exacerbated, compared with LPS individual group. In addition, API promoted caspase-3, Bad mRNA expression and inhibited Bcl-2 mRNA expression in a time-dependent and concentration- dependent manner. Further study found that pro-apoptosis effect of API was related to suppress VDR expression. CONCLUSIONS: API could inhibit the expression of inducible inflammatory factors, therefore exert the strong anti-inflammatory function. However, API could not protect MSC apoptosis induced by LPS but amplified the apoptosis. The apoptosis is related to Bad/Bcl-2 increasing and caspase-3 activation, which is mediated through suppressing VDR expression.