Study On The Effects And Mechanisms Of Baicalin On Alveolar Bone Resorption

The destruction procedures of periodontitis are the interactive results between bacteria plaque and host through complicate molecule mechanisms. Host defense include nonspecific inflammatory reaction and specific immune response (included that humoral immunity, cell immunity and complement system). Periodontitis disease is an Immune-inflammatory disease caused by bacteria. The occurrence and development of periodontitis are concerned with immunologic cell and correlation factors.Frontal resorption is one of the main pathological changes of periodontitis. The aims of periodontitis treatment are to control or eliminate inflammation and to encourage reorganization. Drug control is sine qua non method to eliminate inflammation, block up frontal resorption and promote osteanagenesis. Extractive or monomers of some Chinese medicinal herb were confirmed to be equipped with effect ion of anti-inflammatory and direct or indirect inhibition for differentiation and functional of osteoclast.Baicalin is a flavonoid extracted from the dry root of Labiatae plant Scutellaria. Baicalin is the main indicator component of quality control of Scutellaria and its preparation. According to pharmacological research reports, baicalin has outstanding anti-inflammatory activities and it can inhibit the generation of various kinds factors which encouraged the differentiation and functional of osteoclast. Serial research hint that Baicalin is a promising biological active ingredient for the prevention and cure of periodontitis, but there is still a lot of work on the research of its pharmacological mechanism.During the procedure of periodontitis, the number and activity of T cell rise, so do many inflammatory cell factors, which aggravate the inflammation of periodontium and bone resorption. TGF-βcan suppress the activity and generation of T cells. Then, it is assumed that the mechanism of baicalin's anti-inflammatory and anti- frontal resorption activities were related with TGF-β. TGF-βare mainly secreted by macrophage. So, the presumption of the mechanism of baicalin's anti- frontal resorption activities formed: baicalin may encourage macrophage secret TGF-β, then enhance the inhibition of TGF-βto T cell, causing decrease in the number and activity of T cells, leading to decreased of TNF-αand other inflammatory factors secreted by T-cell under the effect of endotoxin and reduced OPG–RANKL ratio, which further resulted in decreased osteoclast formation and bone loss and the risk factor of periodontitis.The purposes of this study were:①through animal experiment, to study the effect of baicalin on alveolar bone resorption by micro-CT and histological.②To study the effect of baicalin on TGFβ1 in macrophage.③Through RNA interference, RNA of TGFβ1 was silenced. Then, the effects of baicalin on the ratio of RANKL/OPG in HPDL cells were studied. It was confirmed that baicalin diminished the ratio of RANKL/OPG in HPDL cells through TGF-β. Baicalin inhibit the activity, quantity and generation of T cells through TGF-βand then, inflammatory factors secreted by T cell were diminished, further, the ratio of RANKL/OPG in HPDL cells step down. It was revealed the action mechanism of baicalin initially and provided the base in theory and experiment for the prevention and cure of periodontal disease.1. Influence of baicalin on alveolar bone absorption in rats with periodontitisEstablishing the animal model of periodontitis firstly, baicalin were injected repeat at the same time, then, the effects of baicalin on alveolar bone resorption were studied by micro-CT and histological. In this study, by performing ultrahigh-resolution micro-CT scans of alveolar bone, it was confirmed that baicalin could inhibit the alveolar bone absorption induced by Lipopolysaccharide (LPS). Compared with baicalin solution of 0.1μg / ml, baicalin solution of 1.0μg / ml had stronger inhibition.2. Effect of baicalin on TGFβ1 in macrophageMacrophages were cultivated in vitro firstly. Then, under different concentration of baicalin, expression of TGFβ2 in macrophages was detected with RT-PCR and Western-blot. The results showed that baicalin could promote the expression of TGFβ1 in macrophage and the expression of TGFβ2 increased with the rising of baicalin concentration (P <0.05). Baicalin of 1.0μg / ml had the more significant effect.3. Establishment of siRNA Eukaryotic Expression Vectors of Recombinant Targeting Gene TGF-βⅡRReferring to the design principles of siRNA , two siRNA sequences were designed according to the TGF-βRⅡnucleotide sequence reported in GeneBank, and the design requirements of pSilencer3.1H1 vector(Ambion,Inc). TGF-βRⅡ siRNA transcription template was synthesized. Annealing products were connected directly with pSilencer3.1H1 vector which had been digested by BamHⅠand HindⅢbefore. Then, DH5αcompetent cells were transformed by the connected products. The sequencing results are exactly the same as the designed sequence, which means that the siRNA expression vector targeted TGF-βRⅡhas been constructed successfully.4. T cells transfection and identificationT cells that express TGF-βⅡR stablely were transfected by pSilencer3.1H1-TGF-βⅡR(1-2). Unicellular suspension was prepared with monoclonal cell strain of stably transfected siRNA1 and siRNA2. The growth rate of T cells that transfected with siRNA1 and siRNA2 stably are similar to that of normal T cells'in control group, the differences between each group have no statistics significance (P> 0.05). Both the results of RT-PCR and Western blot have shown that the belt of TGF-βRⅡin T cells transfected with siRNA1 is obviously darker than the control zone, while the belt of TGF-βRⅡin T cells transfected with siRNA2 has no significant changes compared with the control group. It illustrates that the expression of TGF-βRⅡin T cells transfected with siRNA1 has been inhibited significantly, which means that the degradation effect of siRNA1 on TGF-βRⅡmRNA in T cells are characteristic and high performance.5. Effect of baicalin on RANKL/OPG in HPDL cellsT cells transfected siRNA1or not together with HPDL cells were place in the medium that had been added with baicalin and LPS. They were divided into six groups and cultured for 48 hours. RT-PCR was used to detect the expression of RANKL and OPG in HPDL cells and observe whether baicalin could affect expression of RANKL-OPG in HPDL cells with and without TGFβsignal transduction. It demonstrated that TGF-βsignaling transduction played an important role in the effect of baicalin on RANKL / OPG ratio in HPDL cells. This study supported that baicalin may enhance the inhibition of TGF-βto T cell, decrease the number or activity of T cells and decreas TNF-αand other inflammatory factors, which are secreted by T-cell under the effect of LPS, then reduced OPG–RANKL ratio, which further resulted in decreasing of osteoclast formation and bone loss. The study also found that baicalin not only act through TGFβto regulate RANKL / OPG ratio in HPDL cells, but also act through other pathway.Conclusion: Baicalin can inhibit alveolar bone absorption effectively. The mechanism of action may be that: baicalin could promote the expression of TGFβ1 in macrophage and then, enhance the inhibition of TGF-βto T cell, decrease the number or activity of T cells and decreas TNF-αand other inflammatory factors (which are secreted by T-cell under the effect of LPS) then reduced OPG–RANKL ratio, which further resulted in decreasing of osteoclast formation and bone loss.