The pulp–dentin complex comprising the soft connective tissue of the dental pulp and the surrounding mineralized dentin—serves as a critical biological barrier that protects the root canal system from microbial invasion. In healthy teeth, the root canal remains sterile. However, disruption of the pulp’s vascular supply—such as from trauma or untreated deep caries—leads to pulp necrosis. In the absence of blood and immune defenses, the root canal becomes an ideal environment for opportunistic bacteria to proliferate. As these pathogens colonize the canal and their toxins diffuse through the apical foramen or dentinal tubules, they provoke a host immune reaction that can result in progressive periapical bone resorption and the infiltration of inflammatory cells into surrounding tissues.

In chronic periapical infections, both protective and destructive immune mechanisms operate simultaneously in a highly orchestrated yet complex manner. Cytokines modulate inflammation and lesion progression, while chemokines and their receptors direct leukocyte trafficking to the infected region, facilitating both defense and repair.

Key T-cell subsets also play pivotal roles:

Th1 and Th17 cells drive lesion progression and periapical bone destruction. Th1 cells release cytokines like IFN‑γ and IL‑12 that activate macrophages, while Th17-derived IL‑17 recruits neutrophils and promotes osteoclastogenesis .

Th2 cells contribute to healing by producing anti-inflammatory cytokines such as IL‑4 and IL‑10, which suppress pro-inflammatory mediators like IL‑1 and TNF‑α and limit tissue damage.

Regulatory T cells (Tregs) act as master regulators, suppressing overactive Th1, Th2, and Th17 responses via immunomodulatory cytokines (especially IL‑10, along with IL‑4 and IL‑8), thereby preserving tissue homeostasis and preventing excessive inflammation.

Furthermore, chemokines such as IL‑8 (CXCL8) and CXCL10 (IP‑10) guide neutrophils and T-cells to the lesion site, coordinating the immune cell influx required for microbial control and initiating reparative responses .

AIM- To study anti and pro inflammatory mediators such as IL-1 beta, IL-4 and TGF- β in symptomatic apical periodontitis.

Eight single-rooted mandibular teeth from Eight patients aged 20–52 years, each presenting with symptomatic apical periodontitis and radiographic periapical radiolucency at Bhabha College of Dental Sciences, Bhopal, were selected for the study. After obtaining informed consent, extraoral and intraoral examinations confirmed the absence of swelling or sinus tracts, and all teeth responded positively to electric and cold pulp testing. Patients who had used systemic antimicrobial or anti-inflammatory medications within the three months prior to sample collection, as well as those with systemic disorders, were excluded from the study. Under strict aseptic conditions, the selected teeth were isolated using a rubber dam, and cleaning and shaping of the root canals were performed with ProTaper NiTi files (Dentsply Maillefer, Ballaigues, Switzerland)  and 5.2% sodium hypochlorite irrigation. On Day 0, post-cleaning samples were collected by inserting three sterile paper points 2 mm beyond the root apex for one minute, cutting them 4 mm from the tip, and placing them in Eppendorf tubes for storage. RNA was then extracted from the periapical interstitial fluid for cytokine/chemokine expression analysis. The experimental group received a calcium hydroxide dressing with chlorhexidine irrigation, applied as a powder/liquid mixture using H-files, while the control group received no dressing. Coronal access cavities were sealed with eugenol-based cement. On Day 7, teeth were reopened, and samples were collected as on Day 0. No clinical signs or symptoms were observed, and final obturation was performed using the vertically compacted thermoplasticized technique.

Total RNA was extracted using the RNeasy® Mini Kit (Qiagen). RNA``` was then stored at − 70 °C. RNA quantity is checked  on  Qubit  flurometer  (Thermofisher #Q33238) using RNA HS assay kit (Thermofisher #Q32851) following manufacturer’s protocol. To measure the purity of the extraction we also  measured  the  RNA concentration on spectrophotometric measurement (Nanodrop 1000, Thermo ScientificTM). All the collected results are provided in the below table.

Table 1: Quality Control Data of Untreated and Treated Samples at Day 0 and 7.

cDNA Systhesis and Expression Analysis:

To ensure the removal of any DNA contamination, all RNA samples underwent further treatment with the RNase-free DNase Set kit (Qiagen). Table 2 provides the details of the primers utilized in this study. Following this, reverse transcription was carried out using the SuperScript III First-Strand Synthesis System (Invitrogen), following the manufacturer’s protocol. After cDNA preparation, the samples were treated with 2 units of RNaseH at 37°C for 20 minutes to degrade any remaining RNA within the RNA-cDNA hybrid.

Table 2: List of Primer Names, Oligonucleotide Sequences, and Corresponding References Used in the Study

To measure the expression of transcription factors (T-bet, GATA3, and FoxP3) and cytokines (IL-1β, IL-4, and TGFβ), reverse transcriptase PCR was performed with 2 μL of template cDNA. The Real-Time Polymerase Chain Reaction (RT-PCR) was conducted under typical conditions using SYBR green assay, beginning with an initial holding phase at 95°C for 10 minutes. This was followed by a cycling phase consisting of 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. The final melting curve phase involved steps at 95°C for 15 seconds, 65°C for 1 minute, and a final step at 95°C for 15 seconds. The relative quantification of the target gene was calculated using the delta-delta Ct method with a reference gene (GAPDH).

A

B

C

D

E

F

Figure 1: RT-PCR analysis showing Ct values represented as peaks and corresponding melting curves for various transcription factors and cytokines. A. T-bet, B. GATA3, C. FoxP3, D. IL-1β, E. IL-4, F. TGF-β. Bold green line depicts the negative controls in all graphs.

 STATISTICAL ANALYSIS

Gene expression levels between study groups were compared using the Mann-Whitney U test. Significance levels are indicated as follows: *-p < 0.05, **-p < 0.01, ***-p < 0.001

Figure 2: Illustration of cytokine expression in untreated (WIM) and treated (IM) patients at day 0 and day 7. A comparison of cytokine gene expression levels between patient groups was performed using the Mann-Whitney U test. Statistical significance is denoted as follows: *-p < 0.05, **-p < 0.01, ***-p < 0.001.

In the control group (without intracanal medicament) the m-RNA expression of IL-1 beta was elevated at day 7 while TGF-beta and IL-4 has not substantially altered at day 7 compared with day 0.

In contrast  (with intracanal medicament) the m-RNA expression of IL-1 beta was reduced at day 7 while TGF-beta and IL-4 has elevated at day 7 compared with day 0.      

This study provides valuable insights into the immunological dynamics of symptomatic apical periodontitis (AP) by examining the expression profiles of key pro-inflammatory (IL-1β) and anti-inflammatory (IL-4, TGF-β) mediators, alongside transcription factors (T-bet, GATA3, FoxP3) associated with T-helper cell subsets. The comparative analysis between treated and untreated groups over a 7-day period offers a nuanced understanding of the host immune response and the modulatory effects of intracanal medication.

Pro-Inflammatory Mediator: IL-1β

IL-1β is a pivotal cytokine in initiating and propagating inflammatory responses, particularly in bone resorption processes associated with AP. The study observed a significant reduction in IL-1β expression in the experimental group after 7 days, indicating the efficacy of calcium hydroxide and chlorhexidine in dampening pro-inflammatory signals. This aligns with existing literature that underscores the role of IL-1β in AP pathogenesis and its modulation through endodontic interventions.

Anti-Inflammatory Mediators: IL-4 and TGF-β

IL-4 and TGF-β are integral to the resolution of inflammation and tissue healing. The experimental group exhibited elevated levels of these cytokines after 7 days, suggesting a shift towards an anti-inflammatory milieu conducive to periapical healing. IL-4, associated with Th2 responses, and TGF-β, linked to regulatory T-cell functions, collectively contribute to suppressing excessive inflammation and promoting tissue repair.

Research on cytokines has highlighted the advantages of using calcium hydroxide and chlorhexidine as root canal dressings, as they enhance anti-inflammatory mediators (such as IL-4 and TGF-β) while reducing proinflammatory mediators (like IL-1) throughout the experimental period.

The findings underscore the therapeutic potential of intracanal medicaments like calcium hydroxide and chlorhexidine in modulating the host immune response. By attenuating pro-inflammatory cytokines and enhancing anti-inflammatory mediators, these treatments can effectively promote periapical healing. Monitoring cytokine profiles could serve as a biomarker for treatment efficacy and disease progression in Apical Periodontitis.

In conclusion, this study highlights the dynamic balance between pro- and anti-inflammatory mediators in Apical Periodontitis and the capacity of endodontic treatments to favourably modulate this balance, paving the way for improved therapeutic strategies. By incorporating these micro- and nanoscale insights into dental practice, we can achieve more effective, minimally invasive, and lasting treatment outcomes that support overall oral health

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