Yunnan Baiyao Might Mitigate Periodontitis Bone Destruction by Inhibiting Autophagy and Promoting Osteoblast Differentiation in vivo, ex vivo and in vitro.

Background and Objective: Periodontitis is an inflammatory disease that eventually destroys tooth-supporting tissue. Yunnan Baiyao (YNBY), a traditional Chinese medicine compound with haemostatic and anti-inflammatory properties has shown therapeutic potential in several diseases. Our previous study revealed that YNBY suppressed osteoclast differentiation in periodontitis. The purpose of this study is to investigate the influences of YNBY on osteoblasts and explore its potential mechanisms. Materials and Methods: A rat periodontitis model was established by ligation of maxillary second molars. After the end of modelling, histopathological observation by hematoxylin-eosin (HE) staining and Masson trichrome staining, detection of bone resorption by Micro-CT scanning, detection of osteoclasts by tartrate-resistant acid phosphatase (TRAP) staining, expression of osteocalcin (OCN) and microtubule-associated protein 1 light chain 3 (LC3) by immunohistochemistry. Lipopolysaccharides was used to irritate MC3T3-E1 osteoblastic cells and ex vivo calvarial organ as an in vitro model of inflammation. CCK-8 assay was performed to examine the toxicity of YNBY to MC3T3-E1 osteoblastic cells. Osteogenesis was assessed with alizarin red staining, immunofluorescence staining, Western blot and immunohistochemical staining. Transmission electron microscopy, fluorescent double staining, Western blot and immunohistochemical staining were employed to detect autophagy. Results: Histological and micro-CT analyses revealed that YNBY gavage reduced bone loss caused by experimental periodontitis and upregulated osteogenic proteins in vivo. YNBY attenuated the production of autophagy-related proteins in periodontitis rats. Additionally, YNBY promoted osteogenesis by inhibiting inflammation-induced autophagy in vitro. Furthermore, YNBY suppressed LPS-mediated bone resorption and promoted the production of osteoblast-related proteins in inflamed calvarial tissues ex vivo. Conclusion: This study demonstrated, through in vivo, in vitro and ex vivo experiments, that YNBY promoted osteoblast differentiation by suppressing autophagy, which markedly alleviated bone destruction caused by periodontitis.

Yunnan Baiyao Inhibits Periodontitis by Suppressing the Autophagic Flux.

BACKGROUND: Yunnan Baiyao (YNBY), a traditional Chinese medicine, is renowned for its anti-inflammatory properties. Recent studies have suggested that YNBY plays a significant role in inhibiting osteoclast differentiation and autophagy, which are essential processes in inflammation and bone resorption associated with periodontitis. However, the precise relationship between autophagy and the mechanism by which YNBY inhibits osteoclastogenesis remains unexplored.The primary objective of this study was to investigate the inhibitory effects of YNBY on the process of osteoclastogenesis and its potential in preventing inflammatory bone loss. METHODS: The animals were subjected to sacrifice at intervals of 2, 4, and 6 weeks postintervention whilst under deep anaesthesia, and specimens were subsequently collected. The specimens were subjected to hematoxylin and eosin (HE) staining, in addition to tartrate-resistant acid phosphatase (TRAP) staining and subsequently imaged employing a digital scanner. The confirmation of osteoclast (OC) differentiation and autophagic flux was achieved through various techniques, including western blotting, transmission electron microscopy (TEM), TRAP staining, pit formation assay, and immunofluorescence. RESULTS: The microcomputed tomography images provided evidence of the effective inhibition of alveolar bone absorption at 2, 4, and 6 weeks following YNBY treatment. Additionally, the histomorphometric evaluations of tissue segments stained with HE and TRAP, which involved measuring the distance between the alveolar bone crest (ABC) and cementoenamel junction (CEJ) and quantifying TRAP-positive OCs, yielded comparable results to those obtained through computed tomography analysis. YNBY treatment resulted in a decrease in the CEJ-ABC distance and inhibition of OC differentiation. Furthermore, in vitro studies showed that the autophagy modulators rapamycin (RAP) and 3-methyladenine (3-MA) significantly affected OC differentiation and function. YNBY attenuated the impact of RAP on the differentiation of OCs, autophagy-related factor activation, and bone resorption. CONCLUSIONS: We hypothesise that YNBY suppresses the differentiation of OC and bone resorption by blocking autophagy. This study reveals that targeting autophagy might be a new alternative treatment methodology for periodontitis treatment.

Identification of Prognostic Markers for Head and NeckSquamous Cell Carcinoma Based on Glycolysis-Related Genes.

Head and neck squamous cell carcinomas (HNSCCs) comprise a heterogeneous group of tumors. Many patients respond differently to treatment and prognosis due to molecular heterogeneity. There is an urgent need to identify novel biomarkers to predict the prognosis of patients with HNSCC. Glycolysis has an important influence on the progress of HNSCC. Therefore, we investigated the prognostic significance of glycolysis-related genes in HNSCC. Our results showed that ELF3, AURKA, and ADH7 of 20 glycolysis-related DEGs were significantly related to survival and were used to construct the risk signature. The risk score showed high accuracy in distinguishing the overall survival (OS) of HNSCC. The Kaplan-Meier curves demonstrated that the risk score was associated with an unfavorable prognosis in patients with female sex, male sex, grade 3, T1/2 stage, N+ stage, N2 stage, M0 stage, and clinical stage III/IV. Independent prognostic analysis showed that clinical stage and risk score were strongly associated with OS. Moreover, the risk score had higher accuracy in predicting 1-, 3-, and 5-year survival. AURKA and ADH7 were only significantly related to M1 macrophages and neutrophils, respectively, while ELF3 was significantly correlated with M2 macrophages and monocytes (all p < 0.05).The ceRNA network demonstrated that miR-335-5p and miR-9-5p may play core roles in the regulation of these three genes in HNSCC. The risk score constructed based on three glycolysis-related genes showed high accuracy in predicting the prognosis and clinicopathological characteristics of HNSCC.

Yunnan Baiyao Ameliorates Rheumatoid Arthritis in Rats by Shifting the Th17/Treg Cell Balance and Preventing Osteoclast Differentiation.

Yunnan Baiyao (YNB) is a traditional Chinese medicine that possesses anti-inflammatory effects. Previously, we have demonstrated the effects of YNB in rheumatoid arthritis (RA) animal models; however, the underlying mechanisms are unclear. In the present study, we aimed to investigate the effects of YNB on the T-helper (Th)17/T-regulatory (Treg) cell balance in a collagen-induced arthritis rat model orally administrated YNB or methotrexate, a widely used therapeutic agent for treating RA. Our results showed that YNB treatment significantly decreased the voix pedis thickness and joint functionality scores and alleviated joint histopathology in these rats. These YNB-induced effects were achieved by decreasing the number of Th17 cells and increasing that of Treg cells in the spleen. Moreover, the interleukin- (IL-) 17 level considerably decreased in the serum of YNB-treated rats, whereas the IL-10 level significantly increased. Furthermore, YNB could inhibit RANKL-induced osteoclast formation by regulating the tumor necrosis factor receptor-associated factor 6/NF-κB/nuclear factor of the activated T-cell pathway. In summary, our study shows that YNB exhibits antiarthritic activity by decreasing the ratio of Th17/Treg cells, regulating the cytokine balance, and inhibiting osteoclast activation, providing an experimental basis that supports the use of this traditional Chinese medicine for the clinical treatment of RA.

Yunnan Baiyao diminishes lipopolysaccharide-induced inflammation in osteoclasts.

Yunnan Baiyao (YNBY) has been refined for hundreds of years and has become a treasure of proprietary Chinese medicine that has significant curative effects in the field of hemostasis, blood circulation, and callus. In past years, YNBY has been demonstrated to play an anti-inflammatory role in bone-related diseases, such as rheumatoid arthritis and osteoporosis. However, the osteoclasts are multinucleated giant cells that resorb bone and participate in the occurrence, development, and progression of these bone-related diseases. Previous studies have reported that the inflammatory function is closely associated with arachidonic acid (AA) metabolism, as well as some inflammatory-related pathways, including the nuclear factor кB (NF-кB), mitogen-activated protein kinase (MAPK), and Wnt5a pathways. Therefore, we speculated that the anti-inflammatory effect of YNBY might be associated with the NF-кB, MAPK, and Wnt5a pathways. In order to further excavate the anti-inflammatory roles of YNBY, lipopolysaccharide (LPS) with an optimal concentration of 1,000 pg/ml was used to induce inflammation in osteoclasts. Our results showed that YNBY with a time- and dose-dependent method decreased the concentration of pro-inflammatory cytokines and the expression levels of cyclooxygenase-1 (COX-1), COX-2, 5-lipoxygenase, and prostaglandin E2. Moreover, it was found that COX-2 was the target gene regulated by YNBY. Finally, using NF-кB and MAPK pathway inhibitors or miRNA101b (involved in the Wnt5a pathway) in tandem with YNBY and the results exhibited that these groups caused a reduction in COX-1 and COX-2 expression, indicating that the anti-inflammatory function of YNBY might directly affect the NF-кB, MAPK, and Wnt5a pathways. PRACTICAL APPLICATIONS: Yunnan Baiyao (YNBY) is mainly extracted from precious Chinese medicines such as Panax notoginseng, borneol, musk, and yam and has a wide range of clinical applications. It is not only used to treat various types of traumatic injuries, but also used for upper gastrointestinal bleeding and wound ulcers, neonatal umbilitis, recurrent oral ulcers, esophagitis, bacterial dysentery, and so on. Although the detailed mechanism of action is not clear at present, it is believed that this is related to its anti-inflammatory, hemostatic, and immune-enhancing effects. Many bone-related diseases, such as rheumatoid arthritis and osteoporosis, are regarded to be intimately related to the inflammatory reaction. Thus, this study aimed to explore the underlying mechanisms of YNBY at anti-inflammatory roles. And our results suggested that YNBY directly affected the inflammatory cytokines and AA metabolic products which referred to the NF-кB, MAPK, and Wnt5a pathways, as well as AA metabolism, respectively. Hence, the practical applications of YNBY are the anti-inflammatory effects used to treat for bone-related diseases.

The anti-inflammatory effects of Yunnan Baiyao are involved in regulation of the phospholipase A2/arachidonic acid metabolites pathways in acute inflammation rat model.

The traditional Chinese medicine Yunnan Baiyao (YNB) has been reported to possess anti­inflammatory properties, however its mechanism of action remains unclear. It was previously reported that YNB ameliorated depression of arachidonic acid (AA) levels in a rat model of collagen-induced arthritis. In the current study, the capacity of YNB to ameliorate inflammation was compared in carrageenan­induced and AA­induced acute inflammation of the rat paw with celecoxib and mizolastine, respectively (n=24 per group). The capacity of YNB to affect the phospholipase A2 (PLA2)/AA pathway (using reverse transcription­quantitative polymerase chain reaction) and release of inflammatory lipid mediators (by ELISA) were investigated. Celecoxib ameliorated carrageenan­induced paw edema, and mizolastine ameliorated AA­induced rat paw edema. YNB alleviated paw edema and inhibited inflammatory cell infiltration in the two models. YNB inhibited production of 5­LOX AA metabolite leukotriene B4 (LTB4), and suppressed expression of 5­LOX, cytosolic PLA2 (cPLA2), 5­LOX­activating protein, and LTB4 receptor mRNA in the AA­induced inflammation model (P<0.05). YNB Inhibited the production of the COX­2 AA metabolite prostaglandin E2 (PGE2) and suppressed expression of COX­2, cPLA2, PGE2 mRNA in the carrageenan­induced inflammation mode (P<0.05). Taken together, the data suggest that modulation of COX and LOX pathways in AA metabolism represent a novel anti-inflammatory mechanism of YNB.

Therapeutic effect of Yunnan Baiyao on rheumatoid arthritis was partially due to regulating arachidonic acid metabolism in osteoblasts.

In order to explore the potential therapeutic effect of Yunnan Baiyao (YNB) on rheumatoid arthritis (RA), rat models were constructed and orally administrated with YNB or methotrexate (MTX) in parallel. Clinical physical, histological and biochemical parameters showed trivial therapeutic difference between YNB and MTX applications. Urine and serum metabonomics results indicated that many endogenous metabolites differentially changed among the rats receiving diverse therapeutic interventions. Among them, the fluctuation of arachidonic acid (AA) was thought to make sense. Thus, its relevant metabolites were subjected to quantitation by using osteoblasts treated by YNB in vitro. It was found that YNB extract of 20 µg/mL could greatly activate the synthesis of intracellular prostaglandin E2 and thromboxane B2 in osteoblasts. Excretion of prostaglandin D2 could be suppressed but not the thromboxane B2. This study proved the efficacy of YNB on curing RA and its potential mechanism through modulating AA metabolism in osteoblasts to some extent.