OBJECTIVES: To explore the promoting effect of ginsenoside Rb3 (Rb3) on osteogenesis in periodontitis environment, and to explain its mechanism. METHODS: Human periodontal ligament stem cells (hPDLSCs) were cultured by tissue block method and identified by flow cytometry. Cell counting kit-8 (CCK8) method and calcein acetoxymethyl ester/propidium iodide staining were used to detect the effect of Rb3 on the viability of hPDLSCs cells. In vitro cell experiments were divided into control group, 10 μg/mL lipopolysaccharides (LPS) group, 10 μg/mL LPS+100 μmol/L Rb3 group and 10 μg/mL LPS+200 μmol/L Rb3 group. Alkaline phosphatase (ALP) staining was used to detect the ALP activity of hPDLSCs in each group after osteogenesis induction. The expression of hPDLSCs interleukin-6 (IL-6), interleukin-8 (IL-8), runt-related transcription factor 2 (RUNX2) and transforming growth factor-β (TGF-β)genes in each group after osteogenesis was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) method. Western blot was used to detect the protein expression of hPDLSCs phosphorrylated extracellular signal-regulated kinase (p-ERK) in each group. Sprague-Dawley rats were randomly divided into the control group, ligation group and ligation+Rb3 group. The left molar-maxillary tissue was subjected to micro-computed tomography (micro-CT) scanning. After the scanning, the left molar-maxilla was made into periodontal tissue sections. Hematoxylin-eosin (HE) staining was used to detect the infiltration and loss of adhesion of inflammatory cells. Masson staining was used to detect the destruction of gingival collagen fibers. Immunofluorescence staining was used to detect the protein expression of RUNX2 and p-ERK. The expression of TGF-β in rat gingival tissue was detected by qRT-PCR. The protein expression of IL-6 in peripheral serum of rats was detected by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to detect the proportion of Treg cells in rat heart blood. The experimental data were statistically analyzed by Graph Pad Prism10.1.2 software. RESULTS: Rb3 had no effect on the cell activity of hPDLSCs. The results of qRT-PCR and ALP staining showed that Rb3 could inhibit the gene expression of IL-6 and IL-8 in inflammatory hPDLSCs, promote TGF-β gene and promote the osteogenic differentiation of inflammatory hPDLSCs. Western blot showed that Rb3 inhibited the protein expression of inflammatory hPDLSCs p-ERK. The results from micro-CT, Masson staining, and HE staining demonstrated that Rb3 promotes alveolar bone formation in rats with periodontitis, while simultaneously inhibiting the destruction of periodontal fibrous tissue, reducing attachment loss, and suppressing inflammatory cell infiltration. The results of flow cytometry showed that Rb3 could promote the differentiation of Treg cells in peripheral blood of periodontitis rats. The results of ELISA and qRT-PCR showed that Rb3 could inhibit the protein expression of IL-6 and promote the gene expression of TGF-β in periodontitis rats. Immunofluorescence results showed that Rb3 could promote the protein expression of RUNX2 and inhibit the protein expression of p-ERK in periodontitis rats. CONCLUSIONS Rb3 can reduce the inflammatory reaction of periodontal tissues in periodontitis rats, and promote the osteogenic differentiation of hPDLSCs by regulating p-ERK pathways.
Animals ; Ginsenosides/pharmacology* ; Osteogenesis/drug effects* ; Periodontitis/metabolism* ; Rats ; Periodontal Ligament/cytology* ; Humans ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Stem Cells/drug effects* ; Interleukin-6/metabolism* ; Rats, Sprague-Dawley ; Interleukin-8/metabolism* ; Cells, Cultured ; MAP Kinase Signaling System/drug effects* ; Transforming Growth Factor beta/metabolism* ; Signal Transduction ; Male ; Phosphorylation ; Lipopolysaccharides ; Extracellular Signal-Regulated MAP Kinases/metabolism* ; Alkaline Phosphatase/metabolism*

OBJECTIVES: This study aimed to explore the therapeutic effect and mechanism of ginsenoside Rb3 on experimental periodontitis and bone resorption in rats. METHODS: Male SD rats were randomly divided into a control group, a ligation group, an Rb3 group, and a doxycycline (Dox) group for in vivo experiments. A periodontitis model was established by ligating the maxillary second molar, and samples were collected after 3 weeks of drug treatment. Micro-CT assessment of alveolar bone resorption was performed, and hematoxylin-eosin (HE) staining was used to observe pathological changes in periodontal and visceral tissues. Tartrate resistant acid phosphatase (TRAP) staining was applied to detect the formation of osteoclasts in periodontal tissues, and enzyme-linked immunosorbent assay (ELISA) was adopted to detect the serum levels of interleukin (IL)-6, IL-8, immunoglobulin (Ig)M, and IgG. Quantitative polymerase chain reaction (qPCR) was employed to detect the expression of factors related to gingival inflammation and osteoclast formation. Immunofluorescence staining was used to detect phospho-extracellular signal-regulated kinase (p-ERK) expression. In vitro experiments were conducted by pretreating RAW264.7 cells with drugs and adding lipopolysaccharides (LPS) stimulation from Porphyromonas gingivalis (P. gingivalis). IL-1β and IL-6 mRNA expression was detected by qPCR, and Western blot was used to detect the effect of Rb3 on the mitogen-activated protein kinases (MAPKs) signaling pathway. RESULTS: Compared with the control group, the ligation group showed significant periodontitis and bone resorption. Compared with the ligation group, the Rb3 group showed a decrease in alveolar bone resorption and osteoclast formation; p-ERK/ERK ratio, IL-1β, IL-6, and nuclear factor of activated T cells (NFATc1) mRNA levels and downstream gene expression in periodontal tissues; serum IL-6, IL-8, IgG, and IgM levels. Rb3 reduced IL-8 and IL-1β mRNA expression levels and p-ERK/ERK and p-p38 MAPK/p38 MAPK ratios in RAW264.7 cells induced by P. gingivalis LPS stimulation. CONCLUSIONS Rb3 inhibits inflammation and bone resorption in experimental periodontitis in rats. Compared with Dox, Rb3 has better effects in inhibiting pro-inflammatory factors and osteoclast gene expression and may exert anti-inflammatory effects by activating the MAPK signaling pathway.
Animals ; Ginsenosides/therapeutic use* ; Rats, Sprague-Dawley ; Male ; Periodontitis/pathology* ; Rats ; Osteoclasts/drug effects* ; Interleukin-1beta/metabolism* ; Interleukin-6/blood* ; Mice ; Alveolar Bone Loss ; Interleukin-8/blood* ; Immunoglobulin G/blood* ; RAW 264.7 Cells ; Transcription Factors

Objective:To investigate the expression of PC4 and SFRS1 interacting protein 1 (PSIP1) in oral squamous cell carcinoma cells and the effects of PSIP1 silencing on the migration and invasion of oral squamous cell carcinoma cells, and to preliminarily explore its mechanism.Methods:The PSIP1 gene of oral squamous cell carcinoma cell line HN30 was silenced by RNA interference technique. HN30 cells were divided into si-NC group (transfected with siRNA-NC) and si-PSIP1 group (transfected with siRNA-PSIP1). Quantitative real-time PCR was used to detect the expression of PSIP1 mRNA. Scratch test and Transwell invasion test were used to detect the migration and invasion abilities of HN30 cells, and Western blotting was used to detect the expression levels of epithelial-mesenchymal transformation (EMT) related proteins in HN30 cells of the two groups.Results:The relative expression levels of PSIP1 of HN30 cells in the si-NC group and si-PSIP1 group were 1.00±0.00 and 0.21±0.06 respectively, with a statistically significant difference ( t=22.30, P=0.002). The scratch healing rates of the si-NC group and si-PSIP1 were (48.21±4.66)% and (42.05±11.74)% at 12 h respectively, with no statistically significant difference ( t=1.46, P=0.173), and the scratch healing rates of the two groups were (86.61±6.06)% and (67.76±3.62)% at 24 h respectively, with a statistically significant difference ( t=8.01, P<0.001). The invasion numbers of HN30 cells in the si-NC group and si-PSIP1 group were 91.00±7.05 and 23.34±4.98, and there was a statistically significantly difference ( t=19.20, P<0.001). Compared with the si-NC group, the migration and invasion abilities of HN30 cells in the si-PSIP1 group decreased significantly (all P<0.001). The expression levels of E-cadherin of the si-NC group and si-PSIP1 group were 1.06±0.02 and 1.43±0.13 respectively, with a statistically significant difference ( t=-4.94, P=0.036), and the expression levels of N-cadherin were 1.00±0.04 and 0.57±0.14 respectively, with a statistically significant difference ( t=5.03, P=0.007). Compared with the si-NC group, the expression level of E-cadherin in the si-PSIP1 group increased, while the expression level of N-cadherin decreased. Conclusion:Silencing the expression of PSIP1 can significantly inhibit the migration and invasion of HN30 cells, and the mechanism may be related to the effect of PSIP1 on the EMT pathway of oral squamous cell carcinoma.

Objective To evaluate the technical method,clinical effect,safety and complication of the endovascular interventional therapy of intracranial A1 segment aneurysms of anterior cerebral artery(ACA).Methods The data of 14 cases with ruptured A1 segment aneurysms received interventional therapy were analyzed retrospectively.All patients were admitted with subarachnoid hem-orrhage (SAH)and classified by Hunt-Hess scale.There were 3 cases of Grade Ⅰ,5 cases of Grade Ⅱ,and 6 cases of Grade Ⅲ. One of fourteen patients was treated by stent implantation alone and 10 patients were treated by coiling alone.The other 3 patients were treated by stent-assisted coiling.Results All the cases were embolized successfully and cured.Angiography immediately after procedure showed Raymond Ⅰ in 1 1 patients,RaymondⅡ in 2 patients and Raymond Ⅲ in 1 patient.In one patient a coil loop was partly left in the parent artery.All of them showed excellent outcome without any serious complication except that one patient suf-fered transient left hemiparesis.Conclusion Endovascular interventional therapy is a safe,effective method in the treatment of the intracranial A1 segment ACA aneurysms.

Objective To reconstruct the initiative procedure of HIV-1 reverse transcription in vitro and establish a methodology of assessing activity of HIV-1 reverse transcriptase (RT) with real-time PCR Methods The tRNALys-3 gene was amplified from genome in healthy individuals through polymerase chain reaction (PCR), and then T7 transcription promoter was added in 5'-terminal of the tRNALys-3. The tRNA[Lys-3 cRNA product was obtained by applying T7 RNA polymerase through a transcription reaction. The 5'-LTR-PBS DNA was also obtained by transcription reaction from the HIV-1 infectious clone and inserted into pGEM-T easy vectors. 5'-LTR-PBS cRNA was obtained by applying SP6 RNA polymerase whose combining site was located in pGEM-T easy vectors. Then the two RNA samples was catalyzed by two kinds of standard reverse transcriptases (SuperScript Ⅲ and HIV-1 standard reverse transcriptase, respectively) and the cDNA was synthesised. The relative activity of RT was determined with the real-time PCR. Results The tRNALys-3 primer and the SP6-5'-LTR-PBS RNA were procured accurately, whose length were 93bp and 872 bp, respectively. After the following serial dilution of Super Script Ⅲ and HIV-1 standard reverse transeriptase:1 : 10, 1: 100, 1:1 000, 1:10 000, each step of reverse transcription process worked successfully. Real-time PCR results showed that Ct values of the two groups were 13.9, 18. 3, 20. 9, 24. 9 and 20. 4, 25. 5, 28. 7, 32. 5 respectively. Conclusion A novel real-time PCR method is developed to assay the RT activity directly through reconstructing the initiation of HIV reproduction, which may be helpful for clinical management, screening of new antiretroviral drugs, and drug resistance test.

Objective To compare postoperative three-dimensional conformal radiotherapy (3DCRT) and conventional radiotherapy (CR) in patients with non-small-cell lung cancer (NSCLC). Methods From Nov. 2002 to Mar. 2006,162 patients with stage ⅠB～ⅢB NSCLC receiving postoperative radiotherapy in our department were restrospetcively analyzed. Among them,86 received 3DCRT and 76 re-ceived CR. The survival outcome, pattern of failure and treatment-related side effects in both groups were an-alyzed. Results The median follow-up was 29.4 months in the 3DCRT group and 24 months in the CR group. The 1-,2- and 3-year local-regional free survival was 97.5% ,83.2% and 83.2% in 3DCRT group, and 84.3% ,76.0% and 65.6% in CR group(χ2= 5.46, P = 0.019), respectively. No statistically signifi-cant difference was found in the overall survival, disease-free survival or distant metastasis-free survival be-tween the two groups. The local-regional failure rate was statistically different between the two groups (14.5% vs 33.3% ,χ2 =7.70,P =0.006). The incidence of distant metastasis in the two groups was simi-lar. Radiation pneumonitis of NCI CTC grade 2-3 occurred in 10 patients(11.6%)in 3DCRT group and 18 (23.7%) in CR group,which was statistically different(χ2 =4.10,P=0.043). Conclusions Postopera-tive 3DCRT for NSCLC provides a better local-regional control and lower incidence of radiation pneumonitis compared with CR.

Objective To determine the prevalence of symmetry of C shaped root canal system in mandibular second molars.MethodsA total of 70 madibular second molars with C shaped root canal system to be considered as needing root canal therapy were included in this study,of which 68 contralateral X ray apical films were obtained.The symmetry of the root and the root canal system was analyzed using magnifying glass.ResultsOf the 68 mandibular second molars with C shaped root canal system diagnosed clinically,62 cases with C shaped root canal system bilaterally,with the prevalence 91.18%.ConclusionThe anatomic feature of mandibular molars with C shaped root canal system is complicated,and the prevalence of symmetry is very high,which is in favor of estimating the anatomic feature from contralateral tooth.