The therapeutic role of baicalein in combating experimental periodontitis with diabetes via Nrf2 antioxidant signaling pathway.

BACKGROUND AND OBJECTIVE: Oxidative stress has been suggested as an important pathogenic factor contributing to chronic periodontitis with diabetes mellitus (CPDM). Previous studies have revealed the potential therapeutic properties of baicalein (BCI) in oxidative stress-related diseases; however, the antioxidant effects of BCI on therapy for individual with CPDM remain largely unexplored. Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a critical role in cellular defence against oxidative stress. In this study, we aim to determine whether BCI prevents diabetes-related periodontal tissue destruction by regulating Nrf2 signaling pathway. MATERIAL AND METHODS: Human gingival epithelial cells (hGECs) were challenged with high glucose (HG, 25 mmol/L) and/or lipopolysaccharide (LPS, 20 µg/mL). Reactive oxygen species (ROS) were detected by fluorescence-activated cell sorting. The changes of antioxidant-related genes, including Nrf2, catalase (Cat), glutamate-cysteine ligase catalytic subunit (Gclc), superoxide dismutase 1 (Sod1), and superoxide dismutase 2 (Sod2), were quantified by real-time PCR. The localization of phospho-Nrf2 (pNrf2, S40) in the nucleus was detected by immunofluorescence staining and laser scanning confocal microscope (LSCM). PNrf2 and total form of Nrf2 were determined using western blot. The above indicators together with mitochondrial membrane potential (MMP) were further investigated in hGECs pre-treated with different concentrations of BCI (0.01, 0.1, or 0.5 µg/mL) before stimulated with HG plus LPS (GP). Finally, the role of BCI in activating Nrf2 signaling pathway and relieving the alveolar bone absorption was examined in the CPDM model of Sprague Dawley rats. CPDM rats were oral gavaged with BCI (50, 100, or 200 mg/kg daily). The pNrf2 was detected by immunohistochemistry, and the alveolar bone absorption was examined by microcomputed tomography. RESULTS: Our results showed that ROS were significantly increased in both groups of HG and LPS, with the strongest generation in the GP group. In terms of ROS-related gene expression, we found that the mRNA levels of Nrf2, Cat, Gclc, Sod1, and Sod2 were significantly decreased in HG and LPS groups. In consistent with the strongest induction of ROS in GP group, the gene expression in GP group was further decreased as compared to those of HG and LPS groups. Also, the expression of pNrf2 exhibited the same trend with the expression of those antioxidant genes. However, the generation of ROS and the loss of mitochondrial membrane potential induced by GP were abolished by pre-treatment with different concentrations of BCI (0.01, 0.1, or 0.5 µg/mL). Interestingly, we observed that BCI promoted the nucleus translocation of pNrf2, as well as the gene expression levels of pNrf2 and its target genes (Cat, Gclc, Sod1, and Sod2). Finally, in the CPDM animal model, we found that BCI (at concentrations: 50, 100, and 200 mg/kg) markedly increased the number of pNrf2-positive cells in periodontal tissue and mitigated the alveolar bone loss. CONCLUSIONS: Our data revealed a potential role for clinic application of BCI under CPDM conditions, suggesting a new therapeutic drug for CPDM patients.

The promotion function of Berberine for osteogenic differentiation of human periodontal ligament stem cells via ERK-FOS pathway mediated by EGFR.

Coptidis Rhizoma binds to the membrane receptors on hPDLSC/CMC, and the active ingredient Berberine (BER) that can be extracted from it may promote the proliferation and osteogenesis of periodontal ligament stem cells (hPDLSC). The membrane receptor that binds with BER on the cell surface of hPDLSC, the mechanism of direct interaction between BER and hPDLSC, and the related signal pathway are not yet clear. In this research, EGFR was screened as the affinity membrane receptor between BER and hPDLSC, through retention on CMC, competition with BER and by using a molecular docking simulation score. At the same time, the MAPK PCR Array was selected to screen the target genes that changed when hPDLSC was simulated by BER. In conclusion, BER may bind to EGFR on the cell membrane of hPDLSC so the intracellular ERK signalling pathways activate, and nuclear-related genes of FOS change, resulting in the effect of osteogenesis on PDLSC.

Repair of human periodontal bone defects by autologous grafting stem cells derived from inflammatory dental pulp tissues.

BACKGROUND: Recently, stem cells derived from inflammatory dental pulp tissues (DPSCs-IPs) have demonstrated regenerative potential, but the real effect remains to be examined. This pilot study attempted to isolate DPSCs-IPs from two patients and to evaluate the feasibility and the effect of reconstructing periodontal intrabone defects in each patient. METHODS: DPSCs-IPs were harvested from two patients with periodontal intrabone defects with their approval. After discussing the biological characteristics of DPSCs-IPs in each patient, DPSCs-IPs were loaded onto the scaffold material ß-tricalcium phosphate and engrafted into the periodontal defect area in the root furcation. After 1, 3, and 9 months, the outcome was evaluated by clinical assessment and radiological study. Furthermore, new samples were collected and the biological characteristics of DPSCs-IPs were further studied compared with normal dental pulp stem cells. The primary cell culture success rate, cell viability, cell cycle analysis, and proliferation index were used to describe the growth state of DPSCs-IPs. In-vitro differentiation ability detection was used to further discuss the stem cell characteristics of DPSCs-IPs. RESULTS: As expected, DPSCs-IPs were able to engraft and had an effect of regeneration of new bones to repair periodontal defects 9 months after surgical reconstruction. Although the success rate of primary cell culture and growth status was slightly inhibited, DPSCs-IPs expressed comparable levels of stem cell markers as well as retaining their multidifferentiation ability. CONCLUSIONS: We developed a standard procedure that is potentially safe and technological for clinical periodontal treatment using human autologous DPSCs-IPs. TRIAL REGISTRATION: According to the editorial policies, the present study is a purely observational study, so trial registration is not required.

Baicalin inhibits toll-like receptor 2/4 expression and downstream signaling in rat experimental periodontitis.

Periodontitis is a severe inflammatory response, leading to characteristic periodontal soft tissue destruction and alveolar bone resorption. Baicalin possesses potent anti-inflammatory activity; however, it is still unclear whether baicalin regulates toll-like receptor (TLR) 2/4 expression and downstream signaling during the process of periodontitis. In this study, the cervical area of the maxillary second molars of rats was ligated and inoculated with Porphyromonas gingivalis (P. gingivalis) for 4weeks to induce periodontitis. Some rats with periodontitis were treated intragastrically with baicalin (50, 100 or 200mg/kg/day) or vehicle for 4weeks. Compared with the sham group, the levels of TLR2, TLR4 and MyD88 expression and the p38 MAPK and NF-κB activation were up-regulated in the experimental periodontitis group (EPG), accompanied by marked alveolar bone loss and severe inflammation. Treatment with 100 or 200mg/kg/day baicalin dramatically reduced the alveolar bone loss, the levels of HMGB1, TNF-α, IL-1ß, and MPO expression, and the numbers of inflammatory infiltrates in the gingival tissues. Importantly, treatment with 100 or 200mg/kg/day baicalin mitigated the periodontitis-up-regulated TLR2, TLR4 and MyD88 expression, and the p38 MAPK and NF-κB activation. Hence, the blockage of the TLR2 and TLR4/MyD88/p38 MAPK/NF-κB signaling by baicalin may contribute to its anti-inflammatory effects in rat model of periodontitis. In conclusion, these novel findings indicate that baicalin inhibits the TLR2 and TLR4 expression and the downstream signaling and mitigates inflammatory responses and the alveolar bone loss in rat experimental periodontitis. Therefore, baicalin may be a potential therapeutic agent for treatment of periodontitis.

[An application of cone-beam CT in the diagnosis of bone defects for chronic periodontitis].

OBJECTIVE: To evaluate the accuracy of cone-beam CT (CBCT) for detection of bone defects in chronic periodontitis and its consistency with periapical film, panoramic radiograph and clinical examination. METHODS: Seventy-five patients with periodontitis were selected in the study. Each patient received clinical examination, periapical film, panoramic radiograph and CBCT examination one week after supragingival scaling. The distance from the alveolar ridge crest to enamelo-cemental junction was measured. The data were statistically analyzed. RESULTS: A total of 1 494 teeth and 8 964 sites were included in the study. Among the three kinds of imaging methods only CBCT could detect the lip (buccal) or tongue (palatine) side of alveolar bone destruction. Compared with panoramic radiographs[mesial: (4.9±0.4) mm, distal: (4.9±0.8) mm] and periapical film [mesial: (5.1±0.6) mm, distal: (5.1±0.8) mm], CBCT [mesial: (5.5±0.4) mm, distal: (5.6±0.8) mm] showed significant differences (P < 0.01) in alveolar bone defect measurements in detecting mesial and distal alveolar bone defect. There was no significant difference between clinical probing [mesial: (5.5±0.6) mm, distal: (5.5±0.6) mm] and CBCT. CONCLUSIONS: CBCT have the highest consistency with clinical probing in detecting the alveolar bone loss in chronic periodontitis.

Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/CMC-online-HPLC/MS.

In present study, an online analytical method using human periodontal ligament cell/cell membrane chromatography (hPDLC/CMC) combined with high-performance liquid chromatography/mass spectrometry (HPLC/MS) was used for direct recognition, separation, and identification of compounds for the first time from Scutellaria baicalensis Georgi (SBG) that are active on hPDLCs. Baicalein (BAI) and wogonin (WOG), which were identified as the active compounds of the ethyl ether extract of S. baicalensis Georgi (SBGEE), could bind to the same membrane receptor of hPDLC for simvastatin (SIM). Moreover, BAI (0.15-0.6 mg/L) and WOG (0.015-0.6 mg/L) had the capability to enhance cell proliferation, matrix calcification, and formation of calcified nodules, which are comparable to the activities of SIM (0.1 mg/L) in vitro. These observations are consistent with the K(A) of the various drugs. It is very important for the development of SBG used to treat periodontitis.

Induction of immune response and prevention of alveolar bone loss with recombinant Porphyromonas gingivalis peptidylarginine deiminase.

OBJECTIVE: Treatments for periodontitis are not absolutely perfect, and a vaccine against Porphyromonas gingivalis (P. gingivalis) could become a valuable adjunct therapy for periodontitis. DESIGN: In this study, a vaccine of peptidylarginine deiminase (PAD) from P. gingivalis was evaluated in P. gingivalis-induced murine lesion and periodontitis models. The prevention of alveolar bone loss analysis determined by micro-computed X-ray tomography (micro-CT), and histological assays. Furthermore, the induction of immune response of mouse anti-PAD done with ELISA and Western Blot analysis. RESULTS: Compared with animal immunization with incomplete Freund's adjuvant (IFA) alone, PAD group significantly inhibited (P<0.05) bone resorption. ELISA and Western Blot showed that PAD induced response involving immunoglobulin G1 (Ig G1) predominantly. CONCLUSIONS: These results suggest that PAD could be a candidate antigen for a vaccine against P. gingivalis infection.

[Preliminary study of the dual release baicalin and rhBMP-2 system to improve periodontal tissue regeneration in minipigs].

PURPOSE: To explore the feasibility of dual release baicalin and rhBMP-2 system for the treatment of periodontal diseases in minipigs. METHODS: Four miniature swines were selected to establish the model of periodontitis and randomly divided them into four groups: Dual release group, hydrogel with baicalin-GMS and rhBMP-2; Single Huang group, hydrogel with baicalin-GMS; BMP group, hydrogel with rhBMP-2; Negative control group, blank hydrogel. All parameters including clinical indicators of periodontitis, the level of IL-1 and TNF-α in the gingival crevicular fluid (GCF) were measured before and 6 weeks after modeling, and 4, 8 weeks after treatment. The data was analyzed using SPSS13.0 software package. The periodontitis model animals were sacrificed at the end of 8 weeks after treatment. The histological changes of periodontal tissues were observed under microscope with HE staining. RESULTS: (1)In all groups, there were significant differences of clinical indicators and levels of IL-1 and TNF-α in the GCF between periodontitis was established and 4, 8 weeks after treatment (P<0.05). Dual release group was significantly superior than that of other three groups (P<0.05). Moreover, all the parameters improved continuously. X-ray showed that bone mineral density and height in the remaining three groups increased compared with the control group, with highest in dual release group. (2)The results of histological observation showed that the inflammation reaction of periodontal tissues in negative control group was more serious than that in other groups 8 weeks after treatment. There was no obvious repair reaction in the control group, but different amount of new bone was seen in other three groups. Among them, dual release group had most obvious repair reaction. CONCLUSIONS: The dual-slow-release chitosan thermosensitive hydrogel system is employable to be used effectively in the regeneration of periodontal defects, which laid a foundation for further clinical use.

Combining human periodontal ligament cell membrane chromatography with online HPLC/MS for screening osteoplastic active compounds from Coptidis Rhizoma.

We have developed an online analytical method that combines human periodontal ligament cell membrane chromatography (hPDLC/CMC) with high-performance liquid chromatography and mass spectrometry (LC/MS) for recognizing and identifying osteoplastic active components from Coptidis Rhizoma. Retention fractions on hPDLC/CMC were enriched onto an enrichment column and the components were directly analyzed by combining a 10-port column switcher with an LC/MS system for separation and preliminary identification. Using simvastatin (SIM) as a positive control, berberine from Coptidis Rhizoma was identified as the active component which could act on the hPDLC. The MTT colorimetric assay, alkaline phosphatase (ALP) activity, and staining tests revealed that berberine could promote hPDLC growth, increase the secretion of ALP in the culture medium, and enhance the formation of mineralized nodule, thus it is a potential osteoplastic ingredient. This hPDLC/CMC-online-LC/MS method can be applied for screening active components acting on hPDLC from traditional Chinese medicines exemplified by Coptidis Rhizoma and will be of great utility in drug discovery using natural medicinal herbs as a source of leading compounds.

[Cloning and expression of peptidylarginine deiminase of Porphyromonas gingivalis].

PURPOSE: To clone the peptidylarginine deiminase(PAD) gene of Porphyromonas gingivalis(P.gingivalis) and to be expressed in E. coli under the best conditions. METHODS: With P.gingivalis strains ATCC33277 genomic DNA as a template, PCR was applied to obtain gene PAD which was then inserted into linear cloning vector PMD-18-T to construct clone recon. Recombinant PMD18-T-PAD was cloned and analyzed with PCR and restriction endonuclease,and PET-28a expression vector was digested by Xhol and Ncol,their products were linked to construct expression plasmid PET-28a-PAD under certain connection system. The recombinant expression plasmid PET-28a-PAD which had been confirmed correctly was transformed to E. coli competent cells BL21 and induce the expression of PAD with IPTG of different density and time. With His Tag monoclonal antibody as the first antibody, the expressed fusion protein was characterized by Western blot. RESULTS: DNA sequencing showed that the fragment was same as the sequence published in NCBI. Under the condition of 37 degrees centigrade, 0.5mmol/L IPTG, 250r/min shaking for 6 hours, the PAD could be highly expressed. CONCLUSIONS: The PAD is successfully cloned and expressed in E. coli which can be further uesd to study the immunity of PAD and the homologues antibody preparation.

[Construction and identification of BIRC5 shRNA lentiviral expression vector].

AIM: Design and synthesis complementary DNA sequences of 3 pairs of short hairpin structure and a pair of negative control sequence by RNA interference technique and construct and identify a lentiviral interference vector with human BIRC5 gene as target gene. METHODS: The designed and synthesised Single-Stranded primer were annealed to Double-Stranded oligo sequences and subcloned into linear pMAGic lentiviral plasmid vector digested by enzyme Age I and EcoR I. Screening positive clone after transformed into DH5α competent cells and identified by PCR amplification and DNA sequencing. RESULTS: 335 bp straps of positive clone and 298 bp straps of negative clone form PCR amplification production have been obtained after gel electrophoresis, the designed and synthesised sequences have been contained in these clone straps confirmed by the result of DNA sequencing. CONCLUSION: Four pairs of BIRC5 shRNA recombinant lentiviral expression vector were constructed successfully, which laid the foundation for researching the inhibition of BIRC5 siRNA target against tumor cells proliferation, induction apoptosis and gene therapy.

[Cloning of the glyceraldehydes 3-phosphate dehydrogenase gene of porphyromonas gingivalis and its expression in E. coli].

OBJECTIVE: To clone the glyceraldehydes 3-phosphate dehydrogenase (GAPDH) gene of Porphyromonas gingivalis (P. gingivalis) and to induce its fusion expression in E. coli. METHODS: GAPDH was obtained by PCR and was inserted into cloning vector pMD-18-T to construct clone recon. Double enzymes digest the recon pMD18-T-GAPDH and the prokaryotic expression vector pET-32a and then connect to get the expressing recon pET-32a-GAPDH. The recombinant expression plasmid which had been confirmed by enzymes digestion was transformed to E. coli competent cells BL21 and induced the expression of GAPDH with isopropyl beta-D-1-thiogalactopyranoside (IPTG) of different density. RESULTS: DNA sequencing showed that the fragment was 99.802% the same to the sequence published in NCBI. Under the best density, IPTG could be highly expressed. CONCLUSION: The GAPDH had been successfully cloned and expressed in E. coli which gets ready for the following experiment to study the immunity of GAPDH and the homologues antibody preparation.

[Experimental study on the functional regulation of naringin in human periodontal ligament cells].

PURPOSE: To detect the effect of naringin on human periodontal ligament cells' (hPDLCs) proliferation,bone formation and OPG mRNA expression. METHODS: hPDLCs were primarily cultured and identified in vitro through enzyme digestion combined tissue culture method. With 3-(4, 5-Dimethylthiazol-2- yl)-2, 5-diphenyltetrazolium bromide (MTT) method, enzyme linked immunosorbent assay and RT-PCR, the hPDLCs' proliferation, alkaline phosphatase (ALP) activity, expression of collagen protein-I and OPG mRNA were observed after treatment with different concentrations (100,10,1.0,0.1,0.01mg/L) of naringin at different times. SPSS16.0 software package was used for statistical analysis. RESULTS: Primary cultured hPDLCs had good shape; Significant promotion of proliferation,ALP activity and collagen protein-I expression of hPDLCs with naringin was found at the dose of 1.0 mg/L; and 1.0mg/L naringin regulated the expression of OPG mRNA in time-dependent manner. CONCLUSION: Naringin might significantly promote the proliferation of hPDLCs and conversion into the osteoblast.

[Analysis of outer membrane proteins in various strains of Porphyromonas gingivalis by surface enhanced laser desorption/ionization time-of-flight mass spectrometry].

OBJECTIVE: To analyze the common outer membrane proteins (OMP) from Porphyromonas gingivalis (Pg) by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and to provide antigens for the subsequent experiments in screening vaccine for periodontitis therapy. METHODS: Four strains of Pg were cultured under anaerobic conditions. The common OMP was extracted through ultracentrifugation and SELDI-TOF-MS was employed to detect the expressions of proteomes by chip H(50). The data was analyzed by Biomarker Wizard. RESULTS: Four kinds of strains of OMP fingerprint spectrum were obtained. Seventy-one proteins of PgATCC33277, 74 proteins of PgW83, 76 proteins of PgW301 and 72 proteins of Pg381 were captured by chip H(50). Thirteen common proteins were identified according to fingerprint spectrum. There was only 1 of the 13 common proteins identified in NCBI protein bank. CONCLUSIONS: SELDI-TOF-MS has good reproducibility and high sensibility and can be used to identify the common OMP of Pg. The 13 proteins have a potential value in the screening vaccine candidate antigen sites for periodontitis.

[Construction of prokaryotic expression vector of FimA gene from Porphyromonas gingivalis, fusion expression and purification in E. coli BL21(DE3)pLyS].

OBJECTIVE: To clone the FimA gene of fimbriae from Porphyromonas gingivalis (P. gingivalis) and to construct prokaryotic expression vector which was induced in E.coli BL21(DE3)pLyS in the form of fusion protein expression and to identify, purify the product of its expression. METHODS: To clone the FimA gene of fimbriae from P. gingivalis and to construct prokaryotic expression vector pET15b-FimA vector which was transformed into the competent cells of BL21(DE3)pLyS. The expression of fusion protein was induced by isopropyl beta-D-1-thiogalactopyranoside (IPTG). With anti-6xHis Tag monoclonal antibody as the first antibody, the expressed fusion protein was characterized by Western blot and purified by Co(2+)-NTA affinity chromatography. RESULTS: Cloned FimA gene sequences and inserted into expression vector of the FimA sequences were related to the sequence in GenBank database showed 100% homology. IPTG induced and then identified by Western blot showed a fragment of 4.1 x 10(4) has been expressed. Co(2+)-NTA affinity chromatography column was used to obtain high concentrations of FimA purified protein. CONCLUSION: The recombinant prokaryotic expression vector of pET15b-FimA was constructed and was expressed and purified successfully in E. coli BL21 (DE3)pLyS. This study laid the experimental foundation to further prepare for monoclonal antibodies of fimbriae of P. gingivalis and to develop the subunit protein vaccine of prevention of periodontitis.

[A preliminary study on screening for Porphyromonas gingivalis outer membrane protein antigen with two-dimensional liquid phase fractionation and matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry].

OBJECTIVE: To screen a variety of Porphyromonas gingivalis (Pg) common outer membrane proteins with two-dimensional liquid phase fractionation (PF2D) and matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) and provide candidate target antigen for the design of vaccines with cross protection against a variety of Pg. METHODS: The outer membrane proteins of Pg301, PgATCC33277 and PgW83 were extracted through ultracentrifugation, and then they were separated by ProteomeLab PF2D protein fractionation system. After separation, the outer membrane proteins were obtained through comparison, and the primary structure of the proteins was identified by MALDI-TOF/TOF-MS and database. RESULTS: Ninety-nine protein samples out of 3 strains of Pg were obtained after the high performance chromato focusing (HPCF) separation process. B7 fractions of 3 strains of Pg were separated by the reversed-phase high performance liquid chromatography (RP-HPCL) separation process. After comparison of peak and retention time of chromatogram, the 8 common protein peaks of 3 strains of Pg were confirmed. The protein samples were identified by MALDI-TOF/TOF-MS, and one of them was known protein arg-gingipain A. CONCLUSIONS: PF2D protein fractionation system is of good reproducibility and high resolution. A combination of PF2D and MALDI-TOF/TOF-MS can be used to identify the common outer membrane proteins of Pg.

[Cloning of the RgpAcd gene of Porphyromonas gingivalis and its expression in E. coli].

OBJECTIVE: To clone the catalytic domain gene sequence of RgpAcd of Porphyromonas gingivalis (P. gingivalis) and to induce its fusion expression in E. coli. METHODS: The desired DNA fragment RgpAcd was obtained by PCR and was separately sequenced and identified by inserting into inter-vector pMD18-T vector. The correctly fragment was linked with and cloned into a prokaryotic expression vector pET-15b. The recombinant expression plasmid which had been confirmed by enzymes digestion was transformed to E. coli competent cells BL21 (DE3) and expression of fusion protein was induced by IPTG. RESULTS: A 1 476 bp specific fragment was obtained and DNA sequencing showed that the fragment was consistent with those of the published. After induction with IPTG, a fusion protein of 5 x 10(4) was visualized on SDS-PAGE gel. CONCLUSION: The protein of RgpAcd will be obtained for further study and its protein was correctly expressed in E. coli BL21 cells.