In TCM, periodontitis belongs to the category of “Tooth Declaration” and “Tooth Exclusion”. According to the “Miscellaneous Works of Ming Medicine”, “Zhi Zhi Fang” and “Pu Ji Fang”, TCM uses dialectical thinking to classify it into kidney-yin deficiency type, qi-blood deficiency type and stomach-fire-up steam type, the last one is a more common type [29, 30]. To this type of periodontitis, TCM often treated with periodontal defeat drink and stomach-clearing soup, both of which are based on raw gypsum and scutellaria and often achieve better results in clinical treatment [29, 31, 32]. Scutellaria baicalensis, as the main ingredient of the two soups mentioned above, is good at “clearing the fire in the upper energizer” and can clear the fire in the heart, lung and stomach, and is now also used in oral inflammatory diseases such as oral ulcer and periodontitis [33]. So far, Scutellaria baicalensis has been developed into a variety of products for oral diseases, such as the compound Scutellaria baicalensis rinse, Shuanghuanglian toothpaste and Shuanghuang tonic preparations [34,35,36,37]. Modern medical research has shown that baicalein, one of the main components of Scutellaria baicalensis, has anti-tumor, anti-bacterial, anti-inflammatory, anti-oxidant and anti-apoptosis effects [38].

Using the topological analysis of the PPI network and the results of GO analysis and KEGG pathways, we hypothesized that the mechanism of baicalein’s treatment for periodontitis might be mainly related to the MAPK, HIF-1, TNF, PI3K-Akt, VEGF and FoxO signaling pathway. It involves many targets such as VEGFA, HIF1A, ESR1, ESR2, MMP2, MMP9, FOS, EGFR, MAPK3, and other cellular biological responses like enzyme-linked receptor protein signaling pathway, response to oxidative stress, and positive regulation of cell migration, kinase binding, RNA polymerase II-specific DNA-binding transcription factor binding and DNA-binding transcription factor binding.

In molecular docking, a binding energy < 0 indicates the ligand molecule can spontaneously bind to the receptor protein, and a binding energy ≤ -5.0 kcal/mol indicates good binding activity between ligand molecule and receptor proteins [39]. It was observed that MMP9 had shown the highest binding energy value of − 6.48 kcal/mol, and the minimum binding energy of baicalein to 7 core targets were ≤ -5.0 kcal/mol, and to most core targets were ≤ -4.0 kcal/mol (Table 3). we also found out that the target proteins and baicalein were bound by hydrogen bonding energy inter molecular force, indicating that baicalein has high binding activity to target proteins such as MMP9, MMP2, ESR1, EGFR, FOS, HIF1A, TP53, which may be the key component of the treatment for periodontitis (Fig. 6).

MMPs are a group of enzymes that degrade the extracellular matrix. MMP-2 and MMP-9 are type IV collagenases, which mainly degrade type IV collagen, gelatin and elastin, and the levels are significantly increased in the gingival tissue and gingival sulcus fluid of patients with periodontitis [40]. The two play a synergistic role in the development of periodontitis and MMP9 is often used as an indicator of periodontal status [41, 42].

EGFR is an epidermal growth factor receptor, and studies have shown that EGFR promotes IL-1β expression and inhibits Smad3 phosphorylation, a mediator of TGF-β1 signaling, leading to inflammation of periodontal tissue and resorption of alveolar bone [43]. TP53 is a cellular tumor antigen. Liu and colleagues [44] have confirmed that in a P.g-LPS induced cellular inflammation model, the enhanced TP53 activity was involved in periodontal inflammatory response by increasing the secretion of IL-1β, IL-6 and TNF-α after leading to cellular redox imbalance and mitochondrial dysfunction.

HIF1A is a hypoxia-inducible factor closely related to chronic inflammation and is an important transcriptional regulator of cells under hypoxic conditions, promoting the glycolytic process by inducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases [45]. Studies have shown that HIF1A downregulates the expression of TNF-α, IL-6, CD86 and the M1/M2 type macrophage ratio, inhibits osteoclast formation, prevents bone resorption and protects periodontal tissue [46, 47]. FOS is a member of the AP-1 family of transcription factors, which is activated in osteoclast precursors and is required for osteoclast differentiation [48]. Studies have shown that mechanical forces induce the upregulation of FOS in periodontal cells, leading to the resorption of alveolar bone [49].

EGFR, FOS, IGF1R, IGF2, MAPK3, TP53, and VEGFA are commonly found in the MAPK signaling pathway. The MAPK signaling pathway family is complex and mainly includes ERK1/2, p38, JNK, and ERK5. It plays an important role in the immune inflammatory and anti-inflammatory response of periodontal tissues, and in the destruction and formation of alveolar bone. Activation of the p38/MAPK signaling pathway has been proved to increase the expression of protein c-Fos in osteoblasts, activate NFATC1, and promote osteoblasts differentiation [50]. Through the ERK1/2 and JNK signaling pathways, IL-1β activates AP-1 to induce MMP-9 expression in osteoblasts and may enhance collagen degradation via MMP-13 or MMP-9 [51], causing periodontal tissue destruction.

EGFR, IGF1R, MAPK3 and VEGFA are commonly found in the PI3K-AKT and HIF-1 signaling pathways. The PI3K/AKT signaling pathway involved in many biological processes, such as cellular inflammation, apoptosis and glucose metabolism [52, 53].Research has found that by activating the PI3K/AKT/Nrf2 signaling pathway, Qianghuo alcohol can inhibit the synthesis of inflammatory mediators such as IL-1β, IL-32, and IL-8 by human gingival fibroblasts under lipopolysaccharide stimulation. At the same time, it can upregulate the expression of antioxidant proteins such as heme oxygenase 1 (HO-1), catalase, and glutathione reductase, inhibit oxidative stress levels, and alleviate periodontal inflammation [54]. Zhao and colleagues [55] found that activating the PI3K/AKT/mTOR signaling pathway via GPR30 could promote the proliferation and osteogenic differentiation in periodontal ligament cells. Park and colleagues [56] proved that schisandrin could induce HO-1 expression in RAW 264.7 cells through activating of signaling pathways such as PI3K/AKT and ERK, downregulate TNF-α and IL-1β, and stimulate the anti-inflammatory effects of macrophages. HIF-1 signaling pathway mainly plays a role in hypoxia-related physiological conditions and pathological processes, including pro-angiogenesis, apoptosis, and inflammation [57, 58]. Studies have found that CoCl2 can promote the expression of IL-1β and MMP-8 through HIF-1 pathway, triggering cellular autophagy after inducing cytotoxicity of periodontal ligament cell [59]. Activation of HIF-1α can also induce the production of MCP-1 and activation of nuclear factor kappa B (NF-κB) in human macrophages, promote the expression of IL-1β, and cause macrophage inflammation and autophagy processes [60].

AKT1, CASP3, FOS, MMP9, MAPK3, and PTGS2 are enriched in the TNF signaling pathway, This pathway mainly involved in immune function and inflammatory response, and is associated with the PI3K/AKT and MAPK signaling pathways. TNF-α, a key protein in the TNF signaling pathway, could upregulate Blimp1 expression by inhibiting PI3K/AKT signaling pathway, promote osteoclastogenesis, and lead to bone resorption [61]. Through NF-kB and p38/MAPK signaling pathways, TNF-α significantly promoted the production of MMP-3 in cementoblasts, which may involve in the degradation or remodeling of periodontal tissue.

Combined with the results of molecular docking, we can speculate that baicalein may act on key signaling pathways like the TNF, PI3K-AKT, HIF-1 and MAPK through core targets such as MMP9, TNF-α, FOS, MAPK3 and AKT1 to inhibit the expression of pro-inflammatory factors and local inflammatory responses, to reduce apoptosis and collagen degradation, and to promote the proliferation and differentiation of osteoblasts for the treatment of periodontal disease.

Macrophages, as an important component of the host defense system, play an important role in tissue destruction and bone resorption. They are also closely associated with the development of periodontitis. As resident cells or monocyte-derived cells recruited after inflammation, phagocytic capacity of macrophages is a key factor in the development of acquired immunity [62]. Macrophages have a high degree of cellular plasticity and are able to respond to different environmental signals. Upon activation, macrophages can differentiate into M1 (classical) or M2 (alternative) phenotype. M1 type has a pro-inflammatory effect manifested by increased secretion of inflammatory cytokines such as TNF-α, MMP-9, IL-6 and MCP1. iNOS is an M1 type macrophage marker. Under the stimulation of inflammatory cytokines such as TNF-α, IL-6 and IL-1β, the body can express iNOS, which can catalyze the synthesis of a large amount of nitric oxide. NO exacerbates autoimmune tissue damage by stimulating the inflammatory response of macrophages and promoting inflammation or cytotoxicity in peripheral cells [63].

In this study, we used P.g-LPS induced macrophages to construct periodontitis model in vitro, and observed the effect of baicalein on periodontitis cell model. Immunofluorescence results showed baicalein down-regulated the expression of iNOS, which indicated the reduction of M1 macrophages. RT-qPCR and ELISA results showed the expression of MMP-9, TNF-α, IL-6 and MCP1 were all significantly reduced both at the mRNA level and the protein level. Through cellular experiments, we could find that 10, 1, and 0.1 µM baicalein inhibited the expression of pro-inflammatory factors and the polarization toward M1 type without affecting macrophage viability, and significantly reduced P.g-LPS induced cellular inflammation.