Heme oxygenase-1: potential therapeutic targets for periodontitis

Carbon monoxide

Carbon monoxide (CO), a small molecule gas, contributes at least 86% of the endogenous carbon monoxide (Ryter, Alam & Choi, 2006). Low concentrations of CO have protective, anti-inflammatory, antioxidant and antibacterial properties (Di Pietro et al., 2020). MAPK are important targets of CO, regulating various important physiological and pathological processes such as cell growth, differentiation, environmental adaptation and inflammatory responses. In rheumatoid arthritis studies, CO was found to exert antioxidant properties by reducing ROS activity through inhibition of NF-κB expression rather than the MAPK pathway (Ryter, Ma & Choi, 2018). However, in the study on gingival fibroblasts, it was found that CO only activated P38 MAPK and had no effect on JNK and ERK expression (Song et al., 2011). Further research is needed to confirm whether CO affects the expression of MAPKs in other periodontal-related cells. Additionally, the anti-inflammatory effects of CO are related to the regulation of TLR4. CO can inhibit the expression of inflammatory factors such as IL-1β, PGE₂ and inducible nitric oxide synthase(iNOS) in periodontitis (Song et al., 2017; Choi et al., 2021). It was found that in diabetic periodontitis, CO could mediate the RAGE/NF-κB pathway to suppress periodontal tissue inflammation (Tian et al., 2024). Reports indicate that CO inhibits osteoclast formation while simultaneously promoting osteogenic differentiation in the context of periodontitis (Song et al., 2017). The Nrf2/HO-1 pathway is another target of CO, which exerts antioxidant effects by activating antioxidant enzymes such as SOD, CAT and GPx (Di Pietro et al., 2020). Moreover, transcription activation factor-3 (STAT-3), phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and HIF-1, are also the targets of CO, which are involved in the regulation of cell protection (Ryter, Ma & Choi, 2018).

Fe²⁺

Free ferrous iron exhibits elevated expression levels during the pathological progression of periodontitis and plays a crucial role in essential physiological activities, including DNA synthesis, ATP synthesis, and oxygen transport (Chen et al., 2022a; Frey & Reed, 2012). However, when excessive storage of ferrous ions occurs, resulting in iron overload, it may initiate the Fenton reaction, catalyzing the generation of ROS and leading to lipid peroxidation and subsequent oxidative damage to tissues (Rehncrona et al., 1982).

HO-1 plays an important role in cytoprotection, effectively helping cells to resist damage. However, concurrently, Fe²⁺, another metabolite of HO-1, has the potential to contribute to the generation of free radicals. This apparent contradiction has long perplexed researchers and has stimulated related studies to explore this phenomenon more deeply. It has been proposed that this may be closely linked to the observation that Fe²⁺ produced by HO-1 activity is associated with elevated levels of ferritin and transferrin (Lanceta et al., 2013; Paiva et al., 2012). These proteins are integral to the pathology of periodontitis, particularly in the regulation of iron metabolism and the mitigation of oxidative stress. The ferroxidase center located within the ferritin heavy chain may further facilitate the oxidation of Fe²⁺ to the relatively less reactive ferric iron (Fe³⁺) (Bou-Abdallah, 2010; Lawson et al., 1989). It has been proposed that HO-1 deficiency may result in disruptions in iron metabolism, consequently leading to reduced transferrin levels (Kartikasari et al., 2009). However, following 3 months of non-periodontal surgical treatment, serum transferrin levels increased significantly, indicating that these proteins are crucial in the management of periodontitis (Shirmohamadi et al., 2016). Furthermore, transferrin receptor 2 has been reported to effectively modulate the inflammatory response in periodontitis, thereby attenuating alveolar bone loss (Lösser et al., 2024). Thus, HO-1 not only safeguards cells from damage but may also mitigate alveolar bone loss induced by periodontitis by regulating the expression of transferrin and the ferritin heavy chain. Therefore, the regulatory relationship between Fe²⁺ produced by HO-1 activity and transferrin as well as the ferritin heavy chain may be of significant importance in the treatment and prevention of periodontitis, warranting careful consideration.

Biliverdin and bilirubin

Biliverdin is produced by HO-1-catalyzed degradation of heme. It can be converted to bilirubin by biliverdin reductase and nicotinamide adenine dinucleotide phosphate (NADPH) (Zhu et al., 2011). Previously, bilirubin was thought to be a hazardous waste product that could lead to liver disease or neonatal jaundice. However, a research in 1954 showed that bilirubin exhibited antioxidant properties that protected vitamin A and unsaturated fatty acids from oxidation (Osiak et al., 2020). Further studies have found that the antioxidant activity of bilirubin increased in the conditions of change from atmospheric oxygen concentration (20%) to tissue oxygen concentration (2%). Moreover, bilirubin could effectively scavenge single-linear oxygen molecules, disrupt free radical chain reactions, and act as a potent antioxidant, surpassing even the antioxidant alpha-tocopherol (Stocker et al., 1987). Low levels of hyperbilirubinemia may reduce the incidence of oxidative stress-related diseases such as cardiovascular disease, diabetes, obesity and metabolic syndrome. For example, it has been shown that topical application of bilirubin promoted healing of diabetic skin wounds by upregulating antioxidant status, promoting angiogenesis and collagen deposition (Zhao et al., 2024). In addition, bilirubin has shown some anti-inflammatory effects, and it was found that bilirubin treatment significantly reduced the expression levels of iNOS, cyclooxygenase-2 (COX-2) and IL-6, which in turn alleviated gastrointestinal inflammation (Nakao et al., 2004). Also, bilirubin treatment could reduce the levels of pro-apoptotic genes and improve the function and survival of allografts. Importantly, in treatments targeting periodontitis, researchers have found a negative correlation between bilirubin serum concentrations and the severity of periodontitis (Chapple, Milward & Dietrich, 2007). Bilirubin, derived from the reduction of biliverdin, is a potent antioxidant that effectively scavenges ROS, thereby preventing both protein and lipid peroxidation (Zhou et al., 2021) (Fig. 1).

Pathophysiological mechanism

The development of periodontitis involves several factors, including inflammation, oxidative stress, apoptosis, pyroptosis and alveolar bone resorption.

Molecular mechanism

Studies have shown that pathological processes such as inflammation, oxidative stress, apoptosis, pyroptosis and alveolar bone resorption in the treatment of periodontitis are closely related to the regulation of HO-1. Therefore, HO-1 has gradually become an important hub in the treatment of periodontitis, attracting great attention from scholars. It has been shown that inducers can regulate HO-1 expression through multiple signaling pathways, which include NF-κB, PI3K-Akt, p38 MAPK and Nrf2, etc., (Park et al., 2011a; Jin et al., 2012; Du et al., 2022).

The NF-κB signaling pathway is activated by ROS, culminating in the upregulation of pro-inflammatory cytokines and chemokines, which ultimately leads to the destruction of periodontal tissues (Özcan et al., 2017). Moreover, numerous studies have demonstrated that CO, a catabolic byproduct of HO-1, can alleviate periodontal inflammation by inhibiting the NF-κB signaling pathway (Song et al., 2017; Choi et al., 2021, 2022). Additionally, the PI3K/Akt pathway is pivotal in the progression of periodontitis, influencing cell proliferation, apoptosis, cytokine secretion and osteoblast differentiation (Liu et al., 2019b). Notably, researchers have revealed that the activation of the PI3K/AKT/HO-1 signaling cascade inhibits periodontal inflammation and imparts significant anti-oxidative stress properties (Park et al., 2011a). On the other hand, the family of MAPKs (including ERK1/2, JNK, and p38) mediates fundamental biological processes and cellular responses in response to hormones, growth factors, cytokines, bacterial antigens and environmental stresses (Souza et al., 2012). JNK has been shown to exert anti-apoptotic effects in response to bacterial invasion, with its activation stimulating the expression of genes associated with resistance to oxidative stress and apoptosis (Wang et al., 2015). Furthermore, several studies have indicated that the activation of specific signaling cascades, such as ERK/HO-1 and JNK/HO-1, can induce an anti-oxidative stress response, thereby effectively suppressing periodontal inflammation (Park et al., 2011a; Jeong et al., 2010).

Nrf2 serves as the principal activator of HO-1. In its resting state, Nrf2 associates with kelch-like ECH-associated protein 1 (Keap1), which is sequestered in the cytoplasm and subjected to degradation via ubiquitination. Under oxidative stress conditions, Keap1 dissociates from Nrf2, resulting in the translocation of Nrf2 to the nucleus, the dissociation of BTB and CNC homology 1 (Bach1) from the HO-1 promoter, and subsequently, the activation of HMOX1 gene expression (which encodes HO-1) (Zhou et al., 2021). For instance, metformin induces the dissociation of Keap1 from Nrf2, thereby enhancing HO-1 expression and exerting a therapeutic effect on diabetic periodontitis (Mohamed Abdelgawad et al., 2021). Furthermore, research has demonstrated that inhibition of the DNA-binding activity of Bach1, coupled with the promotion of its degradation, can lead to the upregulation of HO-1 expression, thereby facilitating periodontal tissue regeneration (Yuan et al., 2024). Beyond the Nrf2/HO-1 signaling mechanism, various other factors influencing HO-1 expression have been extensively investigated. For example, ginsenosides promote HO-1 expression via epidermal growth factor receptor (EGFR), and knockdown of EGFR leads to decreased HO-1 expression, which in turn reverses the anti-inflammatory and osteogenic effects of periodontal tissues (Kim et al., 2021). Additionally, microRNAs are implicated in the regulation of HMOX1 and its upstream genes (e.g., Nrf2, Keap1, Bach1, etc.), offering new avenues for research into the regulation of HO-1.

HO-1 may also play a role in regulating the expression of high mobility group box 1(HMGB1), a highly conserved nuclear protein implicated in the development of various inflammatory diseases, including periodontitis. The release of HMGB1 may be induced under conditions of oxidative stress. However, the activation of HO-1 contributes to the suppression of HMGB1 expression (Yao et al., 2022). In a murine model of neuropathic pain, activation of HO-1 was found to inhibit HMGB1 expression, consequently reducing the levels of pro-inflammatory factors (Chen et al., 2019b). Ha et al. (2012) demonstrated that the HO-1 inhibitor zinc protoporphyrin (ZnPP) exacerbates brain injury by inducing the expression of HMGB1. Additionally, elevated levels of HMGB1 were detected in the gingival sulcus fluid of patients with periodontitis, where HMGB1 induced the expression of pro-inflammatory factors in periodontal cells via activation of the NF-κB pathway (Kim et al., 2010; Luo et al., 2011). Consequently, HMGB1 may serve as a promising downstream target of HO-1 in the context of periodontitis.

In summary, the potential mechanisms underlying the role of HO-1 in periodontitis necessitate further in-depth investigation. By modulating HO-1 and its associated signaling pathways, novel strategies may emerge for the effective treatment of periodontitis. Currently, while gingival sulcus fluid and saliva are readily accessible in clinical settings, there are limited relevant clinical markers available for detecting the onset and progression of periodontitis. HMGB1, as a potential downstream target of HO-1, is anticipated to serve as a significant biomarker for the diagnosis and treatment of periodontitis. However, the majority of studies on periodontitis and biomarkers have been limited to cross-sectional investigations, primarily reporting correlations between biomarkers and periodontitis, while longitudinal studies examining these biomarkers throughout the progression of periodontitis are notably lacking. This limitation represents a significant challenge currently confronting periodontists. For example, it has been demonstrated that HMGB1 is highly expressed in the gingival sulcus fluid of individuals with periodontitis; however, the dynamics of HMGB1 during the progression of periodontitis and in response to HO-1-targeted therapy remain to be fully explored, which will be the focus of our future research. Further investigation into the potential mechanisms of action of HO-1 in periodontitis will pave the way for future personalized treatment strategies utilizing saliva and gingival sulcus fluid. Future studies should concentrate on effectively activating HO-1, mitigating its double-edged sword effect, and elucidating the specific roles of HO-1-related factors, such as HMGB1, in periodontitis. Furthermore, investigating the relationships between additional biomarkers and the development of periodontitis aims to facilitate the development of more effective diagnostic methods and targeted therapies for the prevention and clinical management of periodontitis (Fig. 2).

Natural extracts

In recent years, the anti-inflammatory and antioxidant effects of bioactive substances of plant origin as well as herbal medicines have been extensively studied (Chen et al., 2022b; Gu, Hao & Xiao, 2022). Many natural extracts have been found to exert protective effects on animal models of periodontitis by activating HO-1. HO-1 inducers, such as magnolol, schisandra chinensis α-iso-cubebenol, 6-Shogaol, hesperetin, resveratrol and quercetin, have been investigated for the treatment of periodontitis (Cho & Kim, 2013; Zhao et al., 2021; Liu et al., 2019a, 2021; Park et al., 2011a; Nonaka et al., 2019; Bhattarai et al., 2016).

Clinical drugs

Currently, several clinical drugs have been found to activate HO-1 and exhibit potential value in the treatment of periodontitis. For example, nifedipine, a calcium channel blocker widely used in cardiovascular disease treatment, has been reported to possess anti-inflammatory and antioxidant effects. Nifedipine can alleviate osteoarthritis by activating the Nrf2/HO-1 signaling pathway to counteract oxidative stress caused by free radicals (Yao et al., 2020). In a mouse macrophage environment stimulated by Porphyromonas gingivalis lipopolysaccharide (a periodontal pathogen), nifedipine upregulated HO-1 expression and inhibited the release of inflammatory substances such as NO (Choe et al., 2021). Strontium ranelate is an anti-osteoporosis drug with dual effects of promoting bone formation and inhibiting bone resorption (Yu et al., 2022). Souza et al. (2018) found promotion of HO-1 signaling and inhibition of alveolar bone resorption after ligating the maxillary molars of rats treated with strontium ranelate (100 mg/kg) for 7 days. Dimethyl fumarate (DMF), a drug used for psoriasis and other immune-mediated diseases, weakens intracellular ROS induced by RANKL by enhancing HO-1 expression while inhibiting osteoclastogenesis (Yamaguchi et al., 2018; Matteo et al., 2022). It is speculated that DMF may be a potential inhibitor of bone destruction in periodontitis. However, the specific efficacy of DMF in periodontitis treatment requires further experimental validation.

Additionally, commonly used hypoglycemic agents for diabetes, a prevalent oxidative stress disorder, include biguanides (e.g., metformin) and GLP-1 (glucagon-like peptide-1) receptor agonists. Metformin is regarded as the first-line choice for treating type 2 diabetes, effectively reducing intracellular free radical levels, enhancing insulin sensitivity, and exerting a cytoprotective effect through the upregulation of HO-1 expression (Wang et al., 2022b). Conversely, GLP-1 receptor agonists (e.g., liraglutide) represent an emerging class of antidiabetic drugs that stimulate insulin secretion primarily by mimicking the biological effects of GLP-1. Research has demonstrated that GLP-1 receptor agonists induce HO-1 expression, promote endothelial cell protection and angiogenesis, contribute to diabetic wound healing, and reduce the risk of diabetes-related complications, thereby enhancing the overall prognosis of diabetic patients (Huang et al., 2021c). The pathogenesis of periodontitis, an oxidative stress disorder closely associated with diabetes, involves the interplay between oxidative stress and inflammatory responses. Numerous studies have demonstrated that both biguanides and GLP-1 receptor agonists effectively inhibit the development and progression of periodontitis (Pang et al., 2019; Sawada et al., 2020; Zhang et al., 2020; Neves et al., 2023). Consequently, an in-depth investigation into the specific role of HO-1 in the pathogenesis of periodontitis concerning biguanides and GLP-1 receptor agonists is essential for future treatment and management strategies.

Furthermore, atherosclerosis is an oxidative stress disorder primarily characterized by lipid accumulation within the vessel wall. Research indicates that statins effectively reduce oxidative stress and enhance endothelial function by increasing HO-1 expression levels and inhibiting NADPH oxidase activity, thereby minimizing vascular damage and preserving vascular structural integrity (Piechota-Polanczyk & Jozkowicz, 2017). In a randomized controlled trial concerning periodontitis, scaling and root planing combined with adjunctive statin treatment significantly improved clinical attachment levels and reduced periodontal pocket depth, while effectively decreasing the incidence of bleeding on probing (Alkakhan et al., 2023). Recent studies further suggest that statins possess significant potential for periodontal regeneration, osteoclast modulation and antimicrobial effects in periodontitis (de Carvalho et al., 2021; Parolina de Carvalho et al., 2024). An in-depth study of the mechanism of action of statins in periodontitis, especially the way they exert their effects through the HO-1 pathway, will bring important insights and guidance for the future treatment and management of periodontitis.

In summary, the management of oxidative stress disorders is closely linked to the function of HO-1. Consequently, a thorough investigation into the specific applications of these HO-1-targeted drugs in periodontitis will be vital for future research endeavors. This approach will not only facilitate the enhancement of combination drug strategies for patients with concomitant systemic oxidative stress disorders, thereby avoiding unnecessary duplication of therapy, but is also expected to achieve optimal efficacy and a favorable prognosis. From the perspective of drug regulatory approvals and safety studies, the available clinical drugs that act as HO-1 inducers demonstrate the potential to become effective options for the treatment of periodontitis (Table 2).

Novel HO-1 inducers

In addition to the use of plants, herbs and clinical drugs for HO-1 induction, there has been a recent interest in other modes of obtaining HO-1 inducers for the treatment of periodontitis. Carbon monoxide has been shown to play a significant role in various cellular biological processes, including cell apoptosis and immune-inflammatory responses. Carbon monoxide-releasing molecules (CORMs), as a novel type of compound, can release carbon monoxide in a controlled manner under physiological conditions, thereby increasing the expression of HO-1 in various animal models and cell types (Lv et al., 2020). Choi et al. (2021, 2022, 2015a) demonstrated that lipophilic CORM-2 and water-soluble CORM-3, CORM-401 had inhibitory effects on the inflammatory response induced by Porphyromonas gingivalis lipopolysaccharide through NF-κB inhibition. Additionally, it has been reported that CORM-3 can suppress the expression of the osteoclast factor RANKL and upregulate the expression of osteoprotegerin (OPG), which may have potential therapeutic value in inhibiting bone resorption in periodontitis (Lv et al., 2020). Moreover, vitamin D has been found to have regulatory effects in various inflammatory diseases. ED-71, as an analog of vitamin D, can activate the Nrf2/HO-1 pathway and inhibit the expression of NLRP3, Caspase-1 and IL-1β in gingival fibroblasts under inflammatory conditions, thereby suppressing pyroptosis (Huang et al., 2020). Interestingly, coffee is a common dietary ingredient in everyday life, and opinions differ as to whether it is beneficial or harmful to health. A survey of 16, 730 adults in Korea found that increased coffee intake may further predispose to periodontitis (Han, Hwang & Park, 2016). However, a Japanese cross-sectional study found that coffee consumption during periodontal maintenance treatment was associated with a reduction in the prevalence of severe periodontitis (Machida et al., 2014). As coffee is a complex mixture, different types of coffee have different compositions and different biological effects. This is perhaps why the results of current coffee research are inconsistent (Song et al., 2022). The main components of coffee are caffeine and chlorogenic acid, which are absorbed by the body. Caffeine and chlorogenic acid have been reported to promote the expression of Nrf2 translocation and HO-1, showing antioxidant effect (Huang et al., 2022; Khan et al., 2019). In contrast, a recent study showed that caffeine and chlorogenic acid preparation of artificial coffee promoted AMP-activated protein kinase phosphorylation and reduced the NF-κB pathway to exert anti-inflammatory effects on periodontal cells (Song et al., 2022). However, based on the uncertainty that still exists regarding the toxicity of these synthetic as well as dietary-acquired substances, and what constitutes a safe and effective dosage. Further studies need to be conducted before the substances are ultimately converted into clinical drugs (Tables 3 and 4).

In summary, natural extracts exhibit a reduced incidence of side effects and a lower propensity for resistance compared to their chemical counterparts. Nevertheless, they are confronted with challenges related to extraction purity, potency, and dosage. Polyphenols, terpenoids, isothiocyanates and saponins have been shown to significantly influence various facets of periodontitis via the modulation of HO-1 activity. These compounds have been extensively investigated within the domain of periodontitis. Future research should prioritize the exploration of additional polyphenols, terpenoids, isothiocyanates and saponins, as well as the translation of experimental findings into clinical applications. Furthermore, alkaloids, polysaccharides and quinones exhibit a strong correlation with HO-1, oxidative stress disorders and chronic inflammatory diseases. Nonetheless, there exists a paucity of studies addressing the principal pathways of these compound classes in periodontitis, including HO-1, warranting our sustained long-term research efforts. The adverse effects associated with marketed clinical drugs are more explicitly delineated, and their safety and efficacy are thoroughly documented through clinical trials. The HO-1-targeted clinical drugs employed in the treatment of oxidative stress-related diseases represent promising candidates for the management of periodontitis. Researchers have initiated investigations into novel HO-1 inducers, including synthetic CORM, periostin and compounds derived from dietary vitamins and coffee. However, these innovative HO-1 inducers presently lack adequate clinical evidence to substantiate their efficacy. Consequently, a significant focus of future research will involve the application of acquired clinical data to the development of clinical therapeutics for periodontal disease. Furthermore, it is crucial to persist in investigating the role of HO-1 in periodontitis management as mediated by natural extracts and clinical drugs. This ongoing research will facilitate the development of diverse structures and functional groups to enhance HO-1 expression and the treatment of periodontitis, ultimately leading to the innovation of novel compounds aimed at achieving a comprehensive cure for this condition.