| BackgroundPeriodontitis is a multi-factor chronic infectious disease caused by microorganisms in plaque bioflim.The initiation and development of periodontal disease is through the imbalance of symbiotic oral microflora from dental plaque,which interacts with the host immune defense system,leading to inflammation and disease.Innate immunity is an important first line of defense against microbial invasion.Now it is clear that Toll-like receptors function as key patternrecognition receptors of the innate immune system.They recognize and distinguish highly conserved structures present in large groups of microorganisms and activate innate immune cells through intercellular signal pathways.Lipopolysaccharide(LPS)of porphyromonas gingivalis,the main pathogen of periodontitis,is an important virulence factor in the pathogenesis which can recognized by TLR-2 and TLR-4 in TLRs inducing the activation of intracellular inflammation-related signal pathway,and the release of inflammatory factors,promoting inflammatory response.CORM-3 is a water-soluble transition metal carbon-based compound,which can carry and release CO,to mimic the bioactive activities of CO,such as anti-inflammation,anti-proliferation,protection of cardiomyocytes,reduction of organ transplant rejection,antimicrobial,anti-tumor and so on.The previous studies of our group showed that CORM-3 could effectively down-regulate the expression of TLR-2,TLR-4 mRNA and protein induced by LPS,and the secretion of inflammatory factor IL6,IL-8,and increase the expression of anti-inflammatory factor IL-10,and the inhibitory effect of CORM-3 at 400 μM was the most significant.Based on the above research background,the purpose of this study was to explore the difference of TLR-2,TLR-4 receptor expression in the gingiva of healthy and periodontal disease patients,and to further explore the possible mechanism of the effect of CORM-3 on LPS-induced TLR-2,TLR-4 expression in human periodontal ligament cells in vivo and in vitro.Method:Part I:The expression of TLR-2 and TLR-4 in gingival tissue from patients with chronic periodontitis.From December 2019 to March 2020,20 volunteers were recruited in the Stomatological Hospital of Shandong University.volunteers were composed of 10 healthy volunteers for orthodontic reasons or crown lengthening surgery,gingivotomy and 10 chronic periodontitis patients(The new classification of clinical diagnosis is III stage or Ⅳ stage,B grade.)with chronic periodontitis flap surgery,periodontal abscess incision and other treatment.The researchers introduced the detailed information about the study to the patient,sought the patient’s consent and signed the informed consent form.All volunteers underwent radiographic examination upon entering to the study.The clinical periodontal parameters were assessed at six sites around sampling teeth and included periodontal probing depth(PD),clinical attachment level(CAL),bleeding on probing(BOP)and plaque index(PI)The gingival biopsies were obtained from periodontally healthy subjects as healthy controls,and satisfied the following criteria:1)PD not exceeding 3 mm;2)absence of BOP;3)AL not exceeding 1 mm;and 4)no radiographic evidence of alveolar bone loss.The gingival biopsies obtained from periodontal subjects met the following criteria:1)PD≥4mm;2)BOP(+);3)significant loss of alveolar bone following non-surgical treatment.All the samples were dehydrated,embedded,sliced and immunohistochemical.Observe under a microscope.Each sample to observe and localize the immune response of TLR4 and TLR-2 under the microscope of X20 and X40.Part Ⅱ The effect of CORM-3 on the expression of TLR-2 and TLR-4 in human periodontal ligament cells induced by LPS and the mechanismⅠ)Isolation and culture of human periodontal ligament cellsThe fresh premolars of healthy periodontal patients aged 14-25 years old were extracted for orthodontic needs,and the primary hPDLCs was obtained by periodontal ligament tissue mass culture method.After primary culture to the third generation,the morphology and characteristics were observed under microscope,and the 3-6 generation cells with good growth were selected for follow-up experiment.Ⅱ)Effects of CORM-3 and BAY 11-7082 on the expression of TLR-2 and TLR-4 in human periodontal ligament cells stimulated by LPSCultured human periodontal ligament cells were randomly divided into NC group,LPS group,LPS+CORM-3 group and LPS+BAY 11-7082 group.The normal group was not stimulated.The LPS+CORM3 group was pretreated with 400 μM for 24 hours.The BAY 11-7082 group were pretreated with BAY 11-7082 for 24 hours.Then add 10 μg/ml p.g.LPS into experimental groups for 24 hours.The total mRNA,of each group was extracted and the expression level of TLR-2,TLR-4 gene was detected by RT-qPCR.At the same time,western blot was used to detect the expression level of TLR-2,TLR-4 protein.III)Effects of CORM-3 and BAY 11-7082 on NF-κB pathway in human periodontal ligament cells stimulated by LPSCultured human periodontal ligament cells were randomly divided into NC group,LPS group,LPS+CORM-3 group and LPS+BAY 11-7082 group.The normal group was not stimulated.The LPS+CORM3 group was pretreated with 400 μM for 24 hours.The BAY 11-7082 group were pretreated with BAY 11-7082 for 24 hours.Then add 10 μg/ml p.g.LPS into experimental groups for 24 hours.The total mRNA,of each group was extracted and the expression level of P65,P50,gene was detected by RT-qPCR.At the same time,western blot was used to detect the expression level of P65,p-P65,P50,p-P50 protein.Part III:The effect of CORM-3 on the expression of TLR-2 and TLR-4 periodontal tissue in rats with experimental periodontitis.I)Establishment of experimental periodontitis in ratsTwenty-five Wistar rats,weighing about 200 g,were randomly divided into 5 groups:normal control group,vehicle group,periodontitis group(LPS group),plus drug group(CORM-3 group)and inhibitor group(Bay 11-7082 group).The mice in periodontitis group,drug group and inhibitor group were all ligated bilateral maxillary first molars,and were injected with 1 μg/mL LPS solution 5 μL every other day on the buccal and lingual side,3 times,and fed with sticky food.The rats in the normal control group were intraperitoneally injected with normal saline every other day,the rats in the vehicle group were intraperitoneally injected with 10%DMSO salt solution every two days as vector control(μL/kg),the rats in the treatment group were intraperitoneally injected with CORM-3 solution(10 mg/kg)every other day,and the rats in the inhibitor group were intraperitoneally injected with Bay 11-7082 inhibitor solution(5 mg/kg)every two days.The materials were collected on the 10th day after modeling.Alveolar bone resorption was detected by HE stainingCultured human periodontal ligament cells were randomly divided into two groups normal control group and inhibitor group.The inhibitor group was pretreated with NF-κB pathway inhibitor Bay11-7082 hPDLCs for 24 h,to detect the expression of IκB mRNA and protein in NC group and inhibitor group.Observe under a microscope of X5,X10,X20.II)Effects of CORM-3 and BAY 11-7082 on the expression of TLR-2 and TLR-4 in local inflammatory tissues of rats with experimental periodontitisThe expression of TLR-2,TLR-4,was detected by immunohistochemical staining Immunohistochemical staining was used to detect the expression of TLR-2 and TLR-4 protein in periodontal connective tissue of each group.Ⅲ)Effects of CORM-3 and BAY11-7082 on NF-κB factor in local inflammatory tissue of rats with experimental periodontitisThe expression of NF-κB was detected by Immunofluorescence staining Immunofluorescence staining was used to detect the expression of NF-κB P65 protein in periodontal connective tissue of each groupResults:Part ⅠTLR-4 and TLR-2 expression was detected significantly in both periodontal pocket epithelium and connective tissues in periodontitis group.They were expressed in cell membrane and cytoplasm in epithelial tissue and TLR-4 was mainly observed in connective tissue,which is mainly found in the cytoplasm of vascular endothelial cells,neutrophils,macrophages,plasma cells and fibroblasts.Part ⅡI)The primary hPDLCs,cultured by tissue mass culture method showed that a large number of human periodontal ligament cells dissociated from the tissue mass and grew radially around.The cells showed long fusiform shape,plump shape,uniform cytoplasm,round or oval nucleus and clear nucleoli.After subculture to the third generation,the morphology of the cells was similar to that of the primary generation,and the growth curve was in the shape of "S" and grew well.The cells can be used in follow-up experiments.II)The results of real time quantitative PCR showed that after 24 hours of 10 μg/ml p.g.LPS stimulation,compared with the control group,the expression of TLR-4 gene increased,but the expression of TLR-2 gene had no significant difference.Compared with the experimental group,after pretreatment with CORM-3 and BAY 11-7082,the expression of TLR-2 and TLR-4 genes in hPDLCs stimulated by 10 μg/ml p.g.LPS decreased significantly.After being stimulated with 10 μg/ml p.g.LPS for 24 h,compared with the control group,the expression of hPDLCs P50 and IκB genes was increased,but the expression of p65 gene was not significantly different from that of the control group.Compared in the experimental groups the expression of IκB p65 gene in hPDLCs was significantly decreased after pretreatment with CORM-3 and BAY 11-7082.The results of Western Blot showed that compared with the control group,the expression of TLR-2,TLR-4,p-P65,p-P50,p-IκB was significantly increased after 24 hours stimulation with 10 μg/ml p.g.LPS.In the experimental group,the expression of TLR-2,TLR-4,p-P65,p-P50,P-IκB in hPDLCs stimulated by LPS was significantly decreased after pretreatment with CORM-3 and BAY 11-7082.Part ⅢI)Immunohistochemical staining:the positive expression of TLR-4 and TLR-2 in periodontal ligament tissue of experimental periodontitis model rats was found in blank control group and vehicle group,and increased significantly in inflammation group compared with blank control group and vehicle group.Compared with inflammation group,the positive expression of TLR-4 in CORM-3 group and BAY11-7082 group decreased significantly.II)Immunofluorescence staining:there was only a small amount of NF-κB expression in the blank control group and vehicle group with no significant difference between.Compared with the blank control group and vehicle group,the expression of NF-κB in LPS group was significantly increased.Compared with LPS group,the positive expression of NF-κB was significantly decreased in CORM-3 group and BAY 11-7082 group.Conclusion:1.The expression of TLR-2,TLR-4 in gingiva of patients with chronic periodontitis is higher than that of healthy people,suggesting that TLRs is a potential drug target for the treatment of chronic periodontitis2.Human periodontal ligament cells pretreated with CORM-3 can down-regulate the expression of TLR-2,TLR-4 in hPDLCs induced by LPS and inhibit the signal transduction of NF-κB pathway,while the specific inhibitor of IκB in NF-κB pathway,BAY 11-7082,can achieve the same effect as CORM-3,indicating that CORM-3 may play a role through NF-κB signal pathway.3.Both CORM-3 and BAY 11-7082 can significantly reduce the expression of TLR-2 and TLR-4 in periodontal ligament of periodontitis rats,and inhibit the expression of NF-κB.it is further proved that CORM-3 may act through NF-κB signal pathway in vivo. |
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