Study participants

The study participants included 33 medwakh smokers and 30 non-smokers. The medwakh group smoked more than five times a day on average. All the participants were young adults with ages ranging from 19 to 25. The median age of medwakh smokers and nonsmokers were 24 (3) and 23.5 (1) respectively. Except for smoking history (p < 0.001) and use of interdental aids (p < 0.05), no significant correlation was observed in participant characteristics and age by statistical analysis performed by chi-square test and Mann-Whitney test respectively between medwakh smokers and non-smokers. Similarly, we did not find any significant association when the median age of medwakh smokers (24) and non-smokers (23.5) were compared (p = 0.851); Table 1 [6]).

Table 1 Demographics of the study participants divided into two groups based on smoking status [6]Salivary proteome profile of Medwakh smokers showed significant alterations

Data are available via ProteomeXchange with identifier PXD045901. In the comparative proteomics analysis, 4.7 × 106 spectra were acquired, and 460,873 spectra were identified, yielding 7590 non-redundant peptides, 6672 of which were unique to protein groups identified across 63 saliva samples. Tryptic cleavage efficiency was acceptable with 64% of the identified peptides having no missed cleavage sites (supplementary Fig. 1). The peptide charge distribution revealed a predominance of double and triple charged species as predicted. Using a decoy database strategy and allowing for at most 1% false discovery rate (FDR) at both the peptide and the protein level, MaxQuant analysis of the combined LC-MS/MS data yielded 749 unique protein groups with a median of 6 assigned (minimum 2) and 5 unique (minimum 1) peptides per protein group. Of the 749 protein groups, 422 were retained for quantitative statistical testing after applying a 70% valid value cut-off filter, in which a quantitative value was required for at least 70% of the samples of at least one of the two groups in order to be retained. Of these 422 proteins, 131 were observed to be differentially abundant by t-test (Benjamini-Hochberg multiple testing correction) with a p-value of < 0.05, between the two study groups (supplementary table S1). No samples were omitted and all the study samples (medwakh smoker: N = 33 and non-smoker: N = 30) were considered for the analysis. Protein identification table generated by Maxquant is given in supplementary table S1.

Differential clustering of the salivary proteome profile of medwakh smokers

Principal component analysis (PCA) showed a clear separation in the protein profiles of the study groups based on the smoking status. A well-defined distribution of the proteome profile was observed among medwakh smokers (Fig. 1a). A volcano Plot was further constructed to graphically represent the results of the t-test for the differential level of the unique proteins. Interestingly, among these differentially significant abundant proteins, we found an enhanced abundance for 6 proteins in medwakh smokers and the remaining were significantly less abundant (p < 0.05 and log2 (fold-change) > 1); Table 2; Fig. 1b). Further, hierarchical clustering and heatmap visualization presented more noticeable changes due to medwakh smoking at the protein level. Heat map analysis of the most significantly abundant proteins affected by medwakh smoking revealed the top-ranking proteins as shown in Fig. 1c. Immunoglobulin lambda like polypeptide 5 (IGLL5), Immunoglobulin heavy variable 1–18 (IGHV1-18), Ly6/PLAUR domain-containing protein 3 (LYPD3), Zinc-alpha-2-glycoprotein (AZGP1) and Nucleobindin-1 (NUCB1) were significantly abundant in medwakh smokers. The highly reactive and soluble transglutaminase substrate protein, involucrin (IVL), expressed by keratinocytes of the epidermis was also found to be significantly abundant among the medwakh smoking group. Among the altered key proteins includes those playing key roles in the regulation of immune complex formation (complement factor I, chitinase-3-like protein 1), hemostasis, thrombosis, and oxidative burst (chloride intercellular channel protein 1, neutrophil cytosol factor 4), cell anchorage (F-actin-capping protein subunit alpha-2, Talin-1), nucleotide metabolism (adenylosuccinate synthetase isozyme 2), cellular metabolism (glucose-6-phosphate 1-dehydrogenase, hexokinase-3), pathogen clearance several glycoproteins (proteasome activator complex subunit 2, protein S100-A12, histidine-rich glycoprotein), and cellular proteins degradation (Ubiquitin-like modifier-activating enzyme 1) Table 2).

Fig. 1

(a) Principal component analysis plot of medwakh smokers along with non-smokers. The distribution of proteome profiles of medwakh smokers (green, n = 33) and non-smokers (red, n = 30) are displayed 2-dimensionally based on smoking status. A well-defined distribution in the proteome profile was observed in medwakh smokers in comparison to non-smokers. (b) Volcano plot displaying proteins that altered significantly in medwakh smokers vs. non-smokers. Log2 fold-changed proteins (medwakh smoker/non-smoker) plotted against the -log10 (p-value) highlighting the differentially abundant proteins (two-sample t-test with BH FDR < 0.05). (c) Heatmap with hierarchical clustering of proteomics profile showing relative quantified proteins in the saliva samples of medwakh smokers and non-smokers

Table 2 List of significantly differential abundant proteins highlighting the impact of medwakh smoking on cellular structure and function, inflammatory pathways, oxidative stress, antioxidant responses, and cellular metabolic events in comparison to non-smokersHighlights of the study

The current study highlights several important findings impacted by medwakh smoking. Firstly, salivary levels of certain key immune mediators were found to be significantly altered (less abundant) in medwakh smokers compared to non-smokers, such as complement factor I (CFI) [14], proteasome activator complex subunit 2 (PSME2), calcium-binding protein S100A12, non-enzymatic chitinase-3 like-protein-1 (CHI3L1), histidine-rich glycoprotein (HRG) and non-secretory ribonuclease (RNASE2). In support of this observation, Reactome pathway enrichment analysis identified neutrophil degranulation, innate immune system, interleukin-12 signaling, and immune system as the top enriched pathways in our data. While some of these protein perturbances are observed in cigarette smokers (CHI3L1 [15], PSME2 [16], S100A12 [17] and HRG [18]), the dysregulated levels of CFI, and RNASE2 are specific to medwakh smokers (Fig. 2).

Secondly, the present study noted significant alterations in oxidative stress and antioxidant regulatory proteins in medwakh smokers, like lower abundance in neutrophil cytosol factor 4 (NCF4), endoplasmic reticulum protein 29 (ERP 29), aldo-keto reductase family 1 (AKR1), flavin reductases (NADPH BLVRB), and putative neutrophil cytosol factor 1 (NCF1). The decreased abundance of ERP29, AKR1, and flavin reductases may have negative implications on reducing oxidative burden indicating a potential imbalance between oxidative stress and antioxidant defense mechanisms (Fig. 2). This was again supported by the pathway enrichment data wherein “response to stress” and “response to chemical stress” pathways were significantly enriched.

Fig. 2

Boxplots showing log2 transformed protein levels involved in immune responses, inflammation, oxidative stress and antioxidant responses. Abundance of CFI (Complement factor I), PSME2 (Proteasome activator complex subunit 2), S100A12 (Protein S100-A12), CHI3L1 (Chitinase-3-like protein 1), RNASE2 (Non-secretory ribonuclease), HRG (Heme transporter HRG1), NCF 4 (Neutrophil cytosol factor 4), ERP 29 (Endoplasmic reticulum resident protein 29), AKR1A1 (Aldo-keto reductase family 1 member A1), BLVRB (Flavin reductase (NADPH) and NCF1 (Neutrophil cytosol factor 1) subdivided by medwakh smoking and non-smoking groups

Additionally, an increased abundance of involucrin (IVL), a protein associated with squamous cell differentiation, was observed in medwakh smokers compared to non-smokers. This condition is a common pathological alteration observed in habitual smokers. A rise in abundance of involucrin is reported to result in compromised epidermal permeability barrier [19] eventually leading to increased prevalence of certain cutaneous disorders (Fig. 3). Furthermore, medwakh smoking caused alterations in proteins associated with cellular dynamics including cell anchorage, adhesion and cytoskeletal structure, with all these identified among the top most enriched terms by ClusterProfiler. Tobacco smoking is reported to cause similar alterations in genes and proteins related to metabolism and remodeling of the extracellular matrix [20]. Dysregulated protein identities related to cytoskeletal and extracellular matrix components were significantly less abundant in medwakh smokers, including FERMT3, CAPZA1, ACTN1, and TLN1. These alterations are like those observed in cigarette smokers, where extracellular matrix remodeling can lead to epithelial-mesenchymal transition [21]. Downregulation of CAPZA and Talin-1 levels, associated with hemodynamic stress [22] and altered cell adhesion [23] respectively, further supports these findings. The current study further revealed alterations in the abundance of various enzymes involved in crucial metabolic pathways in medwakh smokers. Notable changes were observed in the abundance of enzymes such as G6PD, HK3, PYGL, and UGP2 (Fig. 3). These findings shed light on the pathological changes in squamous cell differentiation, cytoskeletal proteins, extracellular matrix remodeling, and cell metabolism associated with medwakh smoking.

Fig. 3

Boxplots showing log2 transformed proteins involved in cell differentiation, anchorage, adhesion, and various metabolic processes. Abundance of IVL (Involucrin), FERMT3 (FERM Domain Containing Kindlin 3), CAPZA1 (F-actin-capping protein subunit alpha-1), ACTN1 (Alpha-actinin-1), TLN1 (Talin-1), G6PD (Glucose-6-phosphate 1-dehydrogenase), HK3 (Hexokinase-3), PYGL (Glycogen phosphorylase, liver form) and UGP2 (UTP–glucose-1-phosphate uridylyltransferase) subdivided by medwakh smoking and non-smoking groups

The over-representation analysis (ORA) further strengthens these findings. The ORA highlighted significant alterations in MAPK and ERK signaling cascade, pentose phosphate pathway (PPP), cytoskeletal organization, cellular dynamics, oxidative stress, and immune response alterations. These pathways align closely with the proteomic data, linking the study’s key findings on squamous cell differentiation, oxidative stress, and alterations in immune mediators. Notably, the PPP regulates oxidative stress by producing NADPH, supporting cellular antioxidant needs in a cell-specific manner. The alterations in MAPK and ERK signaling cascade further underscore the impact of oxidative stress responses contributing to cellular damage and apoptosis. Prior studies show acute tobacco exposure activates ERK and p38 MAPK pathways leading to cell death through oxidative stress-induced apoptosis (supplementary Table 2).

To be concise, these findings shed light on the diverse pathological effects of medwakh smoking in the oral cavity, encompassing immune responses, inflammation, oxidative stress, antioxidant defense, squamous cell differentiation and alterations in cell anchorage, adhesion, cytoskeletal proteins, and cell metabolism. The unique dysregulation of CFI and RNASE2 in medwakh smokers and the observed decrease in immune mediators and alterations in oxidative stress-related proteins highlight potential health risks.

Proteomic analysis revealed dysregulation in metabolic, cell adhesion, and cytoskeletal-associated pathways in medwakh smokers

ClusterProfiler (version 4.2.2) revealed the overrepresented canonical pathways of significantly altered proteins in medwakh smokers compared to non-smoking controls. These include pathways related to mitochondrial damage and oxidative burst as evident from the altered expression of HEBP2 and CYFIP Related Rac1 Interactor B, the key players in the collapse of mitochondrial membrane potential before necrotic cell death and oxidative stress responses [24, 25]. The HEBP2 enhances outer and inner mitochondrial membrane permeabilization, especially under conditions of oxidative stress. Other major pathways as revealed in the enrichment analysis are related to cell adhesion, migration, differentiation, and proliferation. These pathways include the expression of proteins such as talins, kindlin-3, filamin A, etc. which are cytoskeletal proteins that are involved in the connections of major cytoskeletal structures to the plasma membrane. These proteins play a significant role in the assembly of actin filaments to membrane glycoproteins thereby contributing to remodeling of the cytoskeletal network to effect changes in cell shape and migration following various stress responses (Fig. 4a).

Joint pathway analysis

To further investigate the impact of medwakh smoking, joint metabolic and proteomics pathways were analyzed using MetaboAnalyst 5.0 version. The 37 differentially expressed metabolites from our prior investigation and 131 differentially expressed proteins were submitted to metaboAnalyst for integration analysis. According to canonical pathway analysis, significantly altered 31 joint pathways among the medwakh smokers were identified and the most relevant ones include glycolysis or gluconeogenesis (p = 1.6714e-06), pentose phosphate (p = 3.4024e-06), fructose and mannose metabolism (p = 0.0025), nicotinate and nicotinamide metabolism (p = 0.0029982), glutathione metabolism (p = 0.0084934), starch and sucrose metabolism (p = 0.024299), amino sugar and nucleotide sugar metabolism (p = 0.027314), purine metabolosim (p = 0.093428) and pyramidine metabolism (p = 0.17742) (Tables 3 and Fig. 4b).

Fig. 4

(a) Over-representation analysis revealed 10 significant pathways. The dots are coded based on the p-value for the enrichment. The dot size denotes the number of significant proteins in each pathway. (b) Joint pathway analysis of top 37 differential metabolites and 131 proteins derived from medwakh smokers in comparison to non-smoking controls. Top enriched pathways included glycolysis or gluconeogenesis, pentose phosphate, fructose and mannose metabolism, nicotinate and nicotinamide metabolism, glutathione metabolism, starch and sucrose metabolism, and amino sugar and nucleotide sugar metabolism

Table 3 Joint analysis pathways of differential proteins and metabolitesMedwakh smoking influences salivary cytokine levels

The performance and survival of immune cells is under strict redox control, influenced by levels of intracellular and extracellular ROS/RNS species [26]. Results from the current study suggests that medwakh smoking may disrupt the delicate balance between oxidative stress and antioxidant defenses, leading to reduced levels of key immune regulators. The findings from the proteomics data were further validated by exploring expression of salivary immune response evaluation panel in medwakh smokers and compared with non-smokers. Out of the 13 cytokines measured in saliva, the levels of Th1 cytokines, (IL-1β (p < 0.05), IL-12p70 (p < 0.05), IL 23 (p < 0.001), IFN-γ (p < 0.01)), Th2 cytokine, IL-6 (p < 0.001) and the chemokine MCP-1 (p < 0.05) were significantly high in medwakh smokers compared to non-smoking controls (Fig. 5). The least abundant salivary cytokine was IFN-γ in non-smokers and in a vast majority of medwakh smokers.

Fig. 5

Levels of cytokines and chemokine (pg/ml) detected in saliva of medwakh smokers (MS) in comparison with non-smokers (NS)