The extracellular polysaccharide inhibit porcine epidemic diarrhea virus with extract and gene editing Lacticaseibacillus.

Lacticaseibacillus is one of the predominant microorganisms in gut from human and animal, and the lacticaseibacillus have effective applications against the viral diarrhea of piglets in the farm. However, the function and the concrete cell single pathways of the active ingredient from lacticaseibacillus was not clear within anti-infection in the postbiotics research. Here, we compared the biological function of extracellular polysaccharides (EPS) purified from lacticaseibacillus casei (L. casei) and gene editing lacticaseibacillus casei with the CRISPER-Cas9 technology, which were with the ability of antioxidation and anti-inflammation, and the EPS could also inhibit the ROS production within the Porcine Small Intestinal Epithelial Cells-J2 (IPEC-J2). Interestingly, we found that both of EPS and genome editing lacticaseibacillus casei could specifically target the IFN-λ expression in the IPEC-J2, which was beneficial against the PEDV infection in the virus replication and production with the qRT-PCR and indirect immunofluorescence methods. Finally, the STAT3 cell single pathway was stimulated to transcribe IFN-λ with the EPS to elucidate the detailed mechanism of activating type III IFN signals receptor of IL-10R2, which play the function between anti-inflammation and anti-virus in the PEDV infection. Taken together, our research linked a postbiotics of EPS with the antiviral infection of PEDV, which suggest that the lacticaseibacillus itself still have displayed the potential immunomodulatory activities, and highlight the immunomodulatory potential of EPS-producing microbes.

The alleviative effect of thyroid hormone on cold stress-induced apotosis via HSP70 and mitochondrial apoptosis signal pathway in bovine Sertoli cells.

Thyroid hormone was involved in gene expression and functional regulation in various signal pathways. Cold stress can increase triiodothyronine (T3) level in the blood. The aim of this study was to investigate the effect of T3 on HSP70 expression and apoptosis in Sertoli cells (SCs) under cold stress in vitro culture at 26 °C, and provide a theoretical and practical basis for improving the reproductive efficiency of bulls in cold areas. SCs were treated with different cold stress duration and different T3 concentrations for pre-screening. HSP70 inhibitor was added later, and the apoptotic rate was measured using flow cytometry. The expression of HSP70 and the main genes of mitochondrial apoptosis pathway were determined by means of real-time PCR and western-blot, respectively. The localization of HSP70 was assessed by immunofluorescence. The results showed that cold stress (26 °C, 6 h) played an inductive role in SCs apoptotic rate (P < 0.01) and the transfer of HSP70 into the nucleus. 100 nM T3 further promoted HSP70 expression and its transfer into the nucleus, which significantly inhibited the expression of vital genes (cyt-c, Caspase-9 and Caspase-3) in mitochondrial pathway (P < 0.05). Subsequently, higher survival and lower apoptotic rates of SCs (P < 0.01) were observed. When T3 and HSP70 inhibitor were added together, the expression of cyt-c, Caspase-9 and Caspase-3 were inhibited (P < 0.05), and then the declining apoptotic rate increased again (P < 0.01). In conclusion, T3 can regulate HSP70 expression and translocation to mediate mitochondrial apoptosis pathway to inhibit SCs apoptosis induced by cold stress.

A haplotype variant of porcine IFIT2 increases poly(I:C)-induced activation of NF-κB and ISRE-binding factors.

Interferon-induced protein with tetratricopeptide repeats (IFIT) 2 is associated with various viral infections and pathogenesis in humans and mice. However, there are few reports on IFIT2 in pigs and the polymorphic information remains unclear. Here, by using a direct PCR sequencing method, we identified four single nucleotide polymorphisms (SNPs), c.259G>A (p.Gly87Ser), c.520T>G (p.Phe174Val), c.571C>T (p.Pro191Ser), and c.879A>G (p.Glu293Glu), for the first time in the coding sequence of the porcine (p) IFIT2 gene from a Chinese local breed (Hebao pig), Western commercial pig breeds (Yorkshire and Landrace), and a Chinese developed breed (Beijing Black pig). SNP c.520T>G (p.Phe174Val) leads to the addition of a tetratricopeptide repeat motif, characteristic structure of the IFIT family. SNPs c.259G>A and c.520T>G are medium polymorphic loci (0.25 < polymorphic information content < 0.5) and distributed differently in Western pig breeds and the Chinese local pig, Hebao, which is well known for its strong resistance to disease. Additionally, they are completely linked. The four SNPs constituted five haplotypes with GTCA and AGCA as dominant. The haplotype variant AGCA, which is mainly present in Hebao pigs, significantly synergized the poly(I:C)-induced activation of transcription factors, including NF-κB and IFN-stimulated response element (ISRE)-binding factors, and the expression of interferon ß, indicating that the variant contributes to the induction or magnitude of the immune response upon viral infection. The data showed that variant AGCA might be useful in improving the resistance of pigs to viruses through marker-assisted selection.

[Effects of Morinda officinalis polysaccharides on FN and FN-EDA of periodontal ligament fibroblasts in inflammatory microenvironment].

PURPOSE: To investigate the effect of Morinda officinalis polysaccharides(MOP) on the expression of fibronectin(FN) and fibronectin containing extra domain A(FN-EDA) in inflammatory periodontal ligament fibroblasts. METHODS: Thirty six rats were randomly divided into a control group(n=12) and a model group (n=24). The model group used orthodontic wire ligation to establish periodontitis. After three weeks, 6 rats from each group were selected and confirmed by Micro-CT to complete the modeling. The remaining rats in the model group were randomly divided into periodontitis group, normal saline(NS) group, and MOP group. In the MOP group, MOP (200 mg/kg for 3 d, 50 µL for 4 weeks) was injected into the palatal side of the left maxillary first molar of the rats. In the NS group, same volume of NS was injected, and no treatment was performed in the periodontitis group. The left maxillary tissue of rats were taken and the pathological changes of periodontal tissue were observed by H-E staining. The expression of FN and FN-EDA was detected by immunohistochemistry. Periodontal ligament fibroblasts were cultured in vitro, the effect of MOP on cell activity detected by CCK-8. The fourth generation cells were divided into control group, inflammation group (10 mg/mL lipopolysaccharide), and experimental group (12.5 µg/mL MOP, 12.5 µg/mL MOP+10 mg/mL lipopolysaccharide). The expression of FN and FN-EDA was detected by qRT-PCR and Western blot. The data were statistically analyzed using Prism 8.0 software package. RESULTS: In vivo experiments, the expression of FN-EDA in the MOP group was significantly lower than that in the periodontitis group and NS group(P＜0.05), and the infiltration of inflammatory cells was reduced. However, there was no significant difference in the expression of FN in each group. In vitro experiments, compared with the control group, the expression of FN-EDA mRNA and protein in the inflammation group was significantly increased(P＜0.000 1). MOP significantly reduced the expression of FN-EDA in inflammatory cells, but had no significant effect on FN expression. CONCLUSIONS: With increased expression of FN-EDA in inflammatory periodontal ligament tissues and cells, MOP may play a role in inhibiting inflammation by down-regulating FN-EDA.

Genome Survey of Male Rana dybowskii to Further Understand the Sex Determination Mechanism.

Rana dybowskii is one of the important aquaculture species in Northeast China. The fallopian tubes of female R. dybowskii are used to prepare oviductus ranae (an important traditional Chinese medicine). Therefore, R. dybowskii females have higher economical value than males. An increasing female R. dybowskii population can increase the benefits from R. dybowskii culture. However, the genome of amphibians is complex, making it difficult to investigate their sex determination mechanism. In this study, we analyzed the genome of male R. dybowskii using next-generation sequencing technology. A total of 200,046,452,400 bp of clean data were obtained, and the K-mer analysis indicated that the depth was 50×. The genome size of R. dybowskii was approximately 3585.05 M, with a heterozygosity rate, repeat sequence ratio, and genome GC content of 1.15%, 68.96%, and approximately 43.0%, respectively. In total, 270,785 contigs and 498 scaffolds were generated. The size of the contigs and scaffolds was 3,748,543,415 and 3,765,862,278 bp, respectively, with the N50 length of 31,988 and 336,385,783. The longest contig and scaffold were of the size 137,967,485 and 1,808,367,828 bp, respectively. The number of contigs and scaffolds > 10K nt was 99,620 and 451, respectively. Through annotation, 40,913 genes were obtained, including 156,609 CDS (i.e., 3.83 CDS per gene). Sequence alignment was performed with the assembled scaffolding genome in this study. Two and one fragment had high homology with two male-specific DNA molecular markers of R. dybowskii discovered previously (namely, MSM-222 and MSM-261, respectively). In addition, the Dmrt1 gene of R. dybowskii was obtained with a length of 18,893 bp by comparison and splicing. The forward primers amplifying MSM-222 and MSM-261 were located at 322-343 and 14,501-14,526 bp of Dmrt1, respectively. However, sequence alignment revealed that MSM-222 and MSM-261 were not located on Dmrt1, and only some homologous parts were observed. This indicated that in addition to Dmrt1, other important genes may play a crucial role in the sex determination mechanism of R. dybowskii. Our study provided a foundation for the subsequent high-quality genome construction and provided important genomic resources for future studies on R. dybowskii.

TRAF-STOP alleviates osteoclastogenesis in periodontitis.

The enhanced osteoclastogenesis contributes to alveolar bone resorption in periodontitis, which increases the risk of tooth loss. To reduce bone destruction, the inhibition of osteoclast development is proposed as a feasible treatment. CD40L-CD40-TRAF6 signal transduction plays a crucial role in inflammation, but how it regulates osteoclast activity in periodontitis has not been elucidated. In this study, we showed the potential role of CD40L-CD40-TRAF6 signaling in periodontitis. CD40L obviously promoted osteoclast formation and bone resorption capacity in vitro. Mechanistically, we found that osteoclastogenesis was enhanced by the overexpression of NFATc1 and NF-κB activation. Importantly, osteoclast activity was effectively suppressed by TRAF-STOP, a small molecular inhibitor of TRAF6. Furthermore, local injection of TRAF-STOP-loaded injectable PLGA-PEG-PLGA hydrogel could alleviate ligation-induced periodontitis in vivo. Taken together, TRAF-STOP shows promising clinical efficacy in periodontitis through alleviating osteoclastogenesis.

Loss in the Antibacterial Ability of a PyrR Gene Regulating Pyrimidine Biosynthesis after Using CRISPR/Cas9-Mediated Knockout for Metabolic Engineering in Lactobacillus casei.

Lactobacillus casei (L. casei) has four possible mechanisms: antimicrobial antagonism, competitional adhesion, immunoregulation, and the inhibition of bacterial toxins. To delineate the metabolic reactions of nucleotides from L. casei that are associated with mechanisms of inhibiting pathogens and immunoregulation, we report that a PyrR-deficient L. casei strain was constructed using the CRISPR-Cas9D10A tool. Furthermore, there were some changes in its basic biological characterization, such as its growth curve, auxotroph, and morphological damage. The metabolic profiles of the supernatant between the PyrR-deficient and wild strains revealed the regulation of the synthesis of genetic material and of certain targeting pathways and metabolites. In addition, the characteristics of the PyrR-deficient strain were significantly altered as it lost the ability to inhibit the growth of pathogens. Moreover, we identified PyrR-regulating pyrimidine biosynthesis, which further improved its internalization and colocalization with macrophages. Evidence shows that the PyrR gene is a key active component in L. casei supernatants for the regulation of pyrimidine biosynthesis against a wide range of pathogens.

Morinda officinalis polysaccharides inhibit the expression and activity of NOD-like receptor thermal protein domain associated protein 3 in inflammatory periodontal ligament cells by upregulating silent information regulator sirtuin 1.

OBJECTIVES: This study aims to investigate the effect of morinda officinalis polysaccharides (MOP) in inflammatory microenvironment on the expression of silent information regulator sirtuin 1 (SIRT1) and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) in periodontal ligament cells. METHODS: Thirty rats were randomly divided into control group (n=6) and model group (n=24). The model group used orthodontic wire ligation to establish periodontitis, and six rats from each group were killed after 3 weeks. The successful modeling was confirmed by Micro-CT. The remaining rats in the model group were randomly divided into natural recovery group, normal saline (NS) group, and MOP group. In the MOP group, MOP [200 mg/(kg·3d), 50 µL for 4 weeks] was injected into the palatal side of the left maxillary first molar of the rats, while the NS group was injected with equal volume of NS. The natural recovery group did not undergo any treatment. The left maxilla tissues of the rats were collected, and pathological changes in perio-dontal ligament cells were observed by hematoxylin-eosin (HE) staining. The expression of SIRT1 and NLRP3 was detected by immunohistochemistry. Cultivate periodontal ligament fibroblasts in vitro and detect the effect of MOP on cell activity using CCK-8. The 4th generation cells were divided into control group, inflammation group (10 µg/mL lipopolysaccharide), and experimental group (5 µmol/L MOP, 5 µmol/L MOP+10 µg/mL lipopolysaccharide). The expression of SIRT1 and NLRP3 was detected by quantitative realtime polymerase chain reaction (qRT-PCR) and Western blot analyses. The acetylation of NLRP3 and the contents of interleukin (IL)-1ß and IL-18 were detected by immunoprecipitation and enzyme-linked immunosorbent assay, respectively. Statistical analysis of data was conducted using Prism 9.0 software. RESULTS: In the vivo experiments, the expression of NLRP3 and SIRT1 in the MOP group decreased significantly compared with that in the natural recovery group and NS group, while the expression of SIRT1 increased (P<0.05) and inflammatory cell infiltration decreased. In the in vitro experiments, the expression of NLRP3 mRNA and protein in the inflammation group increased (P<0.05), while the expression of SIRT1 significantly decreased (P<0.01); MOP upregulated the expression of SIRT1 in inflammatory cells (P<0.05), reduced the expression of NLRP3 and its acetylation level significantly (P<0.05), suppressed the content of IL-1ß and IL-18 in the supernatant (P<0.01). CONCLUSIONS: The SIRT1 expression decreased, and that of NLRP3 expression increased in inflammatory periodontal ligament cells. MOP intervention promoted SIRT1 expression, resulting in the inhibition of NLRP3. Meanwhile, the acetylation level of NLRP3 reduced through deacetylation, leading to the decreased activity of NLRP3. Thus, MOP acted as inflammatory suppressor.

Anti-inflammatory effect of selenium on lead-induced testicular inflammation by inhibiting NLRP3 inflammasome activation in chickens.

Lead (Pb) is a deleterious environmental pollutant that is toxic to testes. Selenium (Se) possesses antioxidant and anti-inflammatory properties. Nucleotide-binding domain, leucine-rich-containing family, pyrin-domain containing-3 (NLRP3) inflammasome is involved in inflammatory response. However, the function of NLRP3 inflammasome in antagonistic effect of Se on inflammation caused by Pb remains unknown. The purpose of this research is to identify anti-inflammatory role of Se on testicular toxicity induced by Pb with an emphasis on oxidative stress, inflammation and NLRP3 signaling pathway in chicken. In present study, sixty seven-day-old Hyline male chickens were assigned into four groups. The feeding program consisted of a commercial diet (0.49 mg/kg Se), a Se-supplemented diet (1 mg/kg Se), a Pb-supplemented diet (0.49 mg/kg Se and 350 mg/kg Pb) and a Se-supplemented and Pb-supplemented diet (1 mg/kg Se and 350 mg/kg Pb), respectively. On the 12th week, blood was collected to measure serum testosterone level and testicular tissues were removed to determine Se and Pb concentrations, testicular function, histological structure, oxidative stress indicators and inflammation-related factors (Nuclear factor-kappaB, tumor necrosis factor-α, cyclooxygenase-2, NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1, interluekin (IL)-1ß, IL-6, IL-18 and interferon-γ). The experimental results showed that after Pb administration, testicular injury was confirmed via histological assessment; testicular dysfunction were further indicated by decreased testosterone level and mRNA expression of steroidogenic acute regulatory protein, cytochrome P450 side-chain cleavage, 3ß-hydroxysteroid dehydrogenase and 17ß-hydroxysteroid dehydrogenase. Moreover, NLRP3 signaling pathway activated by Pb-caused oxidative stress was up-regulated accompanied by promotion in reactive oxygen species, nitric oxide, inducible nitric oxide synthase and malondialdehyde and reduction in antioxidants including glutathione peroxidase and glutathione s-transferase. Se administration ameliorated testicular tissue injury, testicular function, oxidative stress and inflammation. In conclusion, Se exhibited antagonistic role in Pb-induced testicular injury via enhancing antioxidant system and inhibiting inflammation in chickens.

LncRNA CALB2 sponges miR-30b-3p to promote odontoblast differentiation of human dental pulp stem cells via up-regulating RUNX2.

Illuminating the mechanisms of odontoblast differentiation of human dental pulp stem cells (hDPSCs) is the key to find therapeutic clues to promote odontogenesis. LncRNAs play a regulatory role in odontoblast differentiation. Here, we identified a novel lncRNA, named lncRNA CALB2. It was up-regulated in odontoblast-differentiated hDPSCs and potentially interacted with miR-30b-3p and RUNX2. Via gain- and loss-of-function approaches, we found lncRNA CALB2 significantly promoted the odontoblast differentiation of hDPSCs. Then, dual luciferase reporter assay and RNA immunoprecipitation assay revealed that both lncRNA CALB2 and RUNX2 mRNA could directly bind to miR-30b-3p via the same binding sites. Interestingly, miR-30b-3p in hDPSCs was down-regulated and RUNX2 was up-regulated during odontoblast differentiation. Moreover, lncRNA CALB2 knockdown significantly reduced the protein level of RUNX2, DSPP and DMP-1, while miR-30b-3p inhibitor rescued the reduction. Furthermore, miR-30b-3p exerted an inhibitory effect on odontoblast differentiation, which could be reversed by lncRNA CALB2. Collectively, these findings indicate that the newly identified lncRNA CALB2 acts as a miR-30b-3p sponge to regulate RUNX2 expression, thus promoting the odontoblast differentiation of hDPSCs. LncRNA CALB2/miR-30b-3p/RUNX2 axis could be a novel therapeutic target for accelerating odontogenesis.

Bioinformatics analysis to identify the critical genes, microRNAs and long noncoding RNAs in melanoma.

Melanoma, which is usually induced by ultraviolet light exposure and the following DNA damage, is the most dangerous skin cancer. The purpose of the present study was to screen key molecules involved in melanoma.Microarray data of E-MTAB-1862 were downloaded from the ArrayExpress database, which included 21 primary melanoma samples and 11 benign nevus samples. In addition, the RNASeq version 2 and microRNA (miRNA) sequencing data of cutaneous melanoma were downloaded from The Cancer Genome Atlas database. After identifying the differentially expressed genes (DEGs) using Limma package, enrichment analysis and protein-protein interaction (PPI) network analysis were performed separately for them using DAVID software and Cytoscape software. In addition, survival analysis and regulatory network analysis were further performed by log-rank test and Cytoscape software, respectively. Moreover, real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to further verify the expression patterns of several selected DEGs.A total of 382 DEGs were identified in primary melanoma samples, including 206 upregulated genes and 176 downregulated genes. Functional enrichment analysis showed that COL17A1 was enriched in epidermis development. In the PPI network, CXCL8 (degree = 29) and STAT1 (degree = 28) had higher degrees and could interact with each other. Survival analysis showed that 21 DEGs, 55 long noncoding RNAs (lncRNAs) and 32 miRNAs were found to be associated with prognosis. Furthermore, several regulatory relationships were found in the lncRNA-gene regulatory network (such as RP11-361L15.4 targeting COL17A1) and the miRNA-gene regulatory network (such as hsa-miR-375 targeting CCL27 and hsa-miR-375 targeting insulin-like growth factor 1 receptor [IGF1R]). Real-time RT-PCR results showed that the overall direction of differential expression was consistent except COL17A1.CXCL8 interacted with STAT1, CCL27, and IGF1R targeted by hsa-miR-375, and COL17A1 targeted by RP11-361L15.4 might function in the development and progression of melanoma, which should be verified by more detailed experiments.

Selenium for the mitigation of toxicity induced by lead in chicken testes through regulating mRNA expressions of HSPs and selenoproteins.

Lead (Pb) is a toxic element and environmental pollutant. Pb toxicity and antagonistic effect of selenium (Se) on Pb have been deeply studied in mammals. The testis is one of the target organs of Pb in birds. The aim of this study was to investigate the mitigating effect of Se on Pb toxicity in chicken testes by determining messenger RNA (mRNA) expressions of 5 heat shock proteins (HSPs) and 25 selenoproteins. Sixty male chickens (7-day-old) were randomly divided into the control group, the Se group, the Pb group, and the Pb + Se group, and were fed for 90 days. The feeding methods of chickens were as follows: The control group was fed drinking water and commercial diet (0.49 mg/kg Se). Lead acetate was added into the drinking water (350 mg/L Pb). Sodium selenite was added into the commercial diet (1 mg/kg Se). Multivariate correlation analysis and principal component analysis (PCA) were used to define the relationships among all the measured factors and the most important parameters that could be used as key factors, respectively. The results indicated that Se decreased the increase of mRNA expressions of all the HSPs and increased the decrease of mRNA expressions of all the selenoproteins induced by Pb in the chicken testes. HSP70 may be a biomarker of Pb poisoning in the chicken testes. Se alleviated Pb-induced toxicity in the chicken testes through regulating mRNA expressions of HSPs and selenoproteins.

The Role of Transient Receptor Potential Cation Channel, Subfamily C, Member 1 in the Odontoblast-like Differentiation of Human Dental Pulp Cells.

INTRODUCTION: Calcium ions (Ca2+) actively participate in reparative dentin formation by promoting cellular proliferation and differentiation of human dental pulp cells (hDPCs). Transient receptor potential cation channel, subfamily C, member 1 (TRPC1) activates Ca2+ entry upon store depletion in a variety of cell types. However, the function of TRPC1 in hDPCs has not been reported. Therefore, we aimed to analyze the role of TRPC1 in hDPCs undergoing odontoblast-like differentiation. METHODS: Immunohistochemical staining was used to determine the distribution of TRPC1 in pulp tissues. Western blot analysis was used to detect the protein level of TRPC1 in the odontoblast-like differentiation of hDPCs. Knockdown of TRPC1 was performed with an adenoviral vector to evaluate the role of TRPC1 in hDPCs during odontoblast-like differentiation. RESULTS: The results showed that TRPC1 was highly expressed in the cytoplasm of dental pulp cells, especially in the odontoblast layer of the healthy pulp. Moreover, the protein level of TRPC1 increased in a time-dependent manner during the odontoblast-like differentiation of hDPCs. Importantly, knockdown of TRPC1 attenuated the process of odontoblast-like differentiation as indicated by the reduction in mineralized nodules and the down-regulation of dentin sialophosphoprotein and dentin matrix protein 1. Moreover, knockdown of TRPC1 decreased Ca2+ entry to the cytoplasm of hDPCs. CONCLUSIONS: Our data indicated a pivotal role of TRPC1 in the odontoblastlike differentiation of hDPCs, which may be a therapeutic target to enhance reparative dentin formation.

Elevated expression of pleiotrophin in human hypertrophic scars.

Hypertrophic scar (HS) is a cutaneous fibrotic disorder characterized by persistent inflammation, excessive proliferation of fibroblasts, and abundant accumulation of extracellular matrix (ECM) proteins. Pleiotrophin (PTN) is a highly conserved and secreted ECM-associated protein that belongs to a novel family of heparin-binding cytokines with multiple biological functions. The aim of this study was to detect and compare the expression and localization of PTN in HS tissues and normal skin tissues. Surgically removed HS tissue samples and site-matched normal skin specimens were obtained from 18 patients during the scar excision and reconstructive surgery. Semi-quantitative RT-PCR, Western blot analysis and immunohistochemistry were used to determine PTN gene expression and localization in skin tissues. Compared with the normal skin tissues, PTN was highly expressed at both mRNA and protein levels in HS tissues (P < 0.01). In immunohistochemical staining, PTN protein was localized in the cells of both epidermis and dermis in skin tissues, and there were increased staining intensity of PTN in HS tissues than in normal skin samples. In conclusion, elevated expression of PTN is likely to be involved in the pathogenesis of HS. Further studies are still required to elucidate the exact role of PTN in HS formation.

Effect of Enterococcus faecalis lipoteichoic acid on apoptosis in human osteoblast-like cells.

INTRODUCTION: Enterococcus faecalis is commonly detected in persistent apical periodontitis characterized by unimproved periradicular bone resorption. The aim of the present study was to examine the effect of lipoteichoic acid (LTA), a major virulence factor of E. faecalis, on apoptosis of osteoblasts. METHODS: Human osteoblast-like MG63 cells were treated with LTA from E. faecalis at a series of concentrations for 48 hours. The proliferation of the MG63 cells was assessed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5- diphenyl-tetrazolium bromide assay. To examine the apoptosis, the LTA-treated cells were analyzed by flow cytometry by using annexin V-fluorescein isothiocyanate and propidium iodide double staining. Hoechst 33258 staining was performed to observe the morphologic changes of the cells. To investigate the apoptosis at the molecular level, the protein levels of Bcl-2 and Bax were determined by Western blot. Meanwhile, the caspase-3 activity was detected with caspase colorimetric protease assay. RESULTS: The proliferation of MG63 cells was inhibited by E. faecalis LTA in a dose-dependent manner. Flow cytometry assay indicated that LTA had a stimulating effect on MG63 cell apoptosis. Typical morphologies of apoptotic cells were observed under fluorescence microscope. Furthermore, the cell apoptosis was confirmed by (1) the down-regulation of the antiapoptotic protein (Bcl-2), (2) the up-regulation of the proapoptotic protein (Bax), and (3) the elevated caspase-3 activity. CONCLUSIONS: LTA of E. faecalis could inhibit the proliferation and induce apoptosis of human osteoblast-like MG63 cells.

Smad 1/5 is involved in bone morphogenetic protein-2-induced odontoblastic differentiation in human dental pulp cells.

INTRODUCTION: Bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor-ß (TGF-ß) superfamily, which has a broad range of activities that affect many different cell types. Previous research has suggested that BMP-2 induces the differentiation of human dental pulp cells (DPCs) into odontoblast-like cells. However, the mechanism by which BMP-2 induces odontoblastic differentiation has not yet been established. In the present study, we examined the involvement of the BMP/Smad pathway in mediating odontoblastic differentiation in DPCs. METHODS: Levels of phosphorylated and unphosphorylated Smad1/5 were quantified by Western blot analysis in response to BMP-2 and the BMP signaling inhibitor noggin. Some nuclear translocation of Smad1/5 was also observed by immunofluorescence staining in isolated DPCs treated with BMP-2. The effects of noggin on the BMP-2-induced odontoblastic differentiation of DPCs were determined by alkaline phosphatase activity assay, and the expression of odontoblastic markers was evaluated by reverse transcription polymerase chain reaction analysis and Western blotting. RESULTS: We found that BMP-2 induced the phosphorylation and nuclear translocation of Smad 1/5. In addition, noggin significantly inhibited alkaline phosphatase activity and odontoblastic differentiation and reduced the formation of mineralized nodules in BMP-2-treated DPCs. CONCLUSIONS: These findings suggest that Smad 1/5 is involved in BMP-2-induced odontoblastic differentiation in DPCs.

NLRP3 is expressed in human dental pulp cells and tissues.

INTRODUCTION: One of the best-characterized Nod-like receptor (NLR) family members is pyrin domain containing 3 (NLRP3). Intracellular NLRP3 is the most versatile innate immune receptor. On activation, NLRP3 assembles into a multiprotein complex, termed an inflammasome, which regulates the secretion and bioactivity of interleukin-1 family cytokines. NLRP3 has broad specificity for mediating an immune response to a wide range of microbial stimuli or danger signals. Therefore, we hypothesize that NLRP3 plays an essential role in the detection of bacterial pathogens and the initiation of inflammation within the dental pulp. Thus, the aim of this study was to evaluate the expression of NLRP3 in normal human dental pulp cells (HDPCs) and pulp tissues. METHODS: Pulp tissues were collected from freshly extracted human third molars, and HDPCs were prepared from the explants of normal dental pulp tissues. Reverse transcription-polymerase chain reaction and Western blotting were performed to detect the levels of NLRP3 mRNA and protein, respectively. In addition, immunohistochemical staining was used to determine the distribution of NLRP3 in pulp tissues. RESULTS: Normal human dental pulp tissues displayed high levels of NLRP3 mRNA and protein. NLRP3 proteins were principally expressed in odontoblasts and some pulp vascular endothelial cells. Moreover, HDPCs also expressed NLRP3 but at a relatively low level in comparison with that of dental pulp tissues. CONCLUSIONS: The expression of NLRP3 in HDPCs and pulp tissues suggests that NLRP3-mediated signaling pathways may play an important role in dental immune defense.

[The relationship of IL-1beta expressed in buccal cells and the polymorphisms of IL-1beta (+3953) with chronic periodontitis].

PURPOSE: The purpose of this study was to investigate the relationship of IL-1beta expressed in buccal cells and the polymorphisms of IL-1beta(+3953) with chronic periodontitis. METHODS: Thirty-six patients with different severity of periodontal disease and thirty-six subjects with healthy periodontal tissues participated in this study. Buccal cells and peripheral venous blood samples were collected and the periodontal status were recorded. Immunohistochemical analysis of IL-1beta expressed in buccal cells was carried out and the polymorphisms in IL-1beta(+3953) were analyzed by polymerase chain reaction-restriction fragment length polymorphism after extracting DNA from peripheral venous blood. Then the differences in the level of IL-1beta expressed in buccal cells and the distribution of each genotype in IL-1beta(+3953) were compared. Multiple linear regression was used to correlate the periodontal status with the polymorphisms in IL-1beta(+3953) and the expression of IL-1beta) in buccal cells. RESULTS: The carrying ratio of IL-1beta(+3953) allel II in severe chronic periodontitis (12%) was significantly higher than the healthy control group(0). The level of IL-1beta expressed in buccal cells of patients with chronic periodontitis was also significantly higher than that of subjects with healthy periodontal conditions (chi2 = 365.095, P < 0.001). Clinical attachment loss and probing depth correlated positively with the expression of IL-1beta (CAL, P < 0.05; PD, P < 0.01) and no correlation was found between plaque index, bleeding on probing,the polymorphisms in IL-1beta(+3959) and the levels of IL-1beta. CONCLUSIONS: The results of this small sample study suggested that the level of IL-1beta expressed in buccal cells showed the severity of chronic periodontitis and the IL-1beta(+3953) genotype did not make the level of IL-1beta expressed in buccal cells changed significantly.