Changes in dental manifestations in patients with non-alcoholic fatty liver disease and their spouses in the dynamics of clinical observation

Introduction

It has been currently proven that chronic periodontitis (CP) is caused by the combined influence of both endogenous and exogenous risk factors, where metabolic diseases are one of the endogenous factors [1, 2]. Thus, a prominent example is non-alcoholic fatty liver disease (NAFLD), the pathogenesis of which is based on insulin resistance, obesity, and initiation of low-intensity systemic chronic inflammation. These metabolic changes contribute to a decrease in the adaptive periodontal capabilities and conditions are created in the oral cavity for the CP initiation with the subsequent «vicious circle» and comorbid pathology formation [3, 4]. CP is a source of the formation of systemically active pro-inflammatory cytokines, proteolytic enzymes, and reactive oxygen species having a genotoxic effect and contributing to cell death against the background of telomere shortening [5].

Exogenous factors are understood to be a certain usual mode of life characteristic of each person, but against the background of comorbid pathology, some habits become factors of initiation or maintenance of the disease course. Bad habits support the CP course being a source of chronic septicemia and systemically active pro-inflammatory cytokines in the oral cavity [6]. It should be noted that the usual mode of life is subconsciously fixed and coincides, especially at the married couple level, and therefore its analysis and correction should be carried out for spouses simultaneously.

Chronic inflammatory process and oxidative stress negatively affect the length of telomeres of gingival epithelial cells contributing to their premature aging [7]. The life cycle of any eukaryotic human body cell is controlled and depends on specialized DNA-protein structures localized at the chromosome ends. Such structures are called telomeres. Their length gradually decreases with each cell mitosis, and upon completion of repetitions with reaching the Hayflick limit, apoptosis occurs. This is how the physiological cycle of many cells passes, with some exceptions, the end of which is biological aging with phenotype change and natural death.

However, the reduction in telomere length is not only caused by the physiological process — mitosis, but also under the influence of various pathological factors with genotoxic effect. Such changes reduce the barrier gingival epithelium capabilities and increase the oral mucosa permeability for various pathogens, xenobiotics into the internal environment and certainly the negative impact on the NAFLD course.

Research objectives: research the follow-up changes in the periodontium during the correction of endogenous and exogenous risk factors at the level of the spouses.

Object and methods of the research

The research adhered to measures for patient’s health safety, respect for his/her rights, human dignity and moral principles according with principles of the World Medical Association Declaration of Helsinki, Ethical Principles for Medical Research Involving Human Subjects, the Council of Europe Convention on Human Rights and Biomedicine, the Council for International Organizations of Medical Sciences, the International Code of Medical Ethics and relevant legal acts regulating clinical studies.

The research included 44 spouses among which one of the spouses had NAFLD determined according to the recommendations of international and domestic criteria (anthropometric measurement data taking into account the past medical history, clinical and biochemical blood tests, liver ultrasound examination, elastography, steatography and liver steatometry). The condition that the other spouse had no signs of liver disease, but had common feeding and hygiene habits, was the criterion for selecting spouses. The previously obtained data of the control group (25 somatically healthy persons following a healthy lifestyle and being representative according to gender and age) were taken for control.

All patients were interviewed with regard to bad habits (smoking), frequent consumption of sweetened beverages and sugary foods. Also, all patients were thoroughly asked about the usual nature of home oral care (use of personal hygiene items and products, frequency of tooth brushing, etc.).

The amount of epithelial attachment loss, probing depth of the gingival sulcus or periodontal pocket, bleeding while probing were clinically studies with PBI index calculation. The index assessment of the oral hygiene level was made according to the OHI-S index. In order to facilitate the dental health analysis, orthopantomography or computed tomography data were taken into account.

The sensitivity of the tongue taste receptors was determined by the gustometry methodology using 6 glucose solution dilutions with the following concentrations: 0.5%, 1.0%, 1.5%, 2.0%, 2.5% and 5.0%. According to the methodolo­gy, a few solution drops were applied with a pipette to the sweet-sensitive tongue areas, starting with 0.5%. The solution concentration was gradually increased until the patient began to clearly feel the sweetness. The patient rinsed the oral cavity with water during the test breaks. The lowest solution concentration, when the patient felt the sweetness, was taken as the threshold concentration.

Determination of the relative length of telomeres of gingival epithelial cells was performed by PCR method with real-time fluorescence detection using the following methodology: 2 scrapings were taken from the surface of the attached gums with disposable applicators. DNA was isolated from the gingival epithelium by applying reagent kits using relative quantification. The DNA concentration in the samples was measured by fluorometric method on a «Qubit 3.0» fluorometer («Life Technologies», USA) using «Qubit dsDNA HS Assay Kits» («Life Technologies», USA). DNA samples were diluted to a concentration of 2–4 ng/μl and kept until amplification at –20° C. Invitrogen primers («Thermo Fisher Scientific») were used for amplification of telomeric sequences.

During the same visit, each patient was offered an individual treatment-and-prophylactic plan comprising the professional oral hygiene; elimination of traumatic occlusion; individual and paired classes with spouses in oral hygiene care according to the individual hygiene algorithm with the selection of hygiene items and products for motivation and formation of stable skills. All patients were interviewed as for eliminating harmful risk factors for the occurrence of inflammatory periodontal lesions.

Statistical calculations were carried out using the SPSS program by non-parametric method. Medians (Me), interquartile range (first and third quartile — Q1, Q3) were calculated. When determining statistical differences between two independent groups, the Mann — Whitney test was used. When comparing three or more groups, the Kruskal — Wallis test was used. The statistical hypothesis of the odds ratio equality of one (ω=1) was verified using the χ² test. The linear relationship between the indicators was estimated using the Pearson and Spearman coefficients.

Results

Physical status of patients

For NAFLD patients, an increase in body mass index (BMI), clinically observed in the form of excess weight, was characteristic. This indicator was 34.7 kg/m² in the main group indicating the first-degree obesity, but there were also patients with the third-degree obesity. Excess adipose tissue is a source of pro-inflammatory cytokines confirmed by an increase in the level of the blood highly sensitive inflammation marker — C-reactive protein (CRP). The patients with metabolic-associated disease, the CRP level was more than 2 times higher compared to the permissible rate characteristic of the chronic low-intensity inflammation in the body, which, as a rule, has no clinical manifestations. An important pathogenetic component in case of NAFLD is insulin resistance and its objective HOMA-IR indicator almost 2 times exceeded the upper reference level in patients. Accordingly, the insulin content in the blood of NAFLD patients was at the level of the upper reference concentration of 21.69 μU/ml, also objectively informing in favor of insulin resistance (Table 1).

Table 1. Indicators of metabolic disorders in patients of the study groups

Each NAFLD patient had a spouse not having metabo­lic diseases, but attracting a scientific research interest for comparison. It has been determined that the average BMI in the group of spouses with NAFLD was equal to 23.9 kg/m². This exceeded the similar indicator from the control group (22.4 kg/m²) and critically approached the upper reference level. It should be added that in this group comprises persons with an initial-degree obesity. However, HOMA-IR already exceeded the permissible level of 2.9, and the insulin level, although it was within the reference fluctuations on an empty stomach — 15.2 μU/ml, exceeded the range of favorable concentrations (5.5–10 μU/ml). The CRP value allowed to assume the appearance in the body of a possible source of chronic inflammation. All of the above-mentioned indicators were within normal limits in the control group.

Dental status of patients

The most common disease, observed in 61.3% of patients in the main group, was second-degree CP, 22.7% suffered from the first-degree CP, and 15.9% of those examined had no chronic inflammation in the periodontium. The CP clinic certainly had objective confirmation on the part of special dental indices. Thus, the oral hygiene index (OHI-S) demonstrated an unsatisfactory level. The papillary bleeding index (PBI) corresponded to the second degree — linear bleeding after gingival sulcus probing. The average depth of pathological pockets was equal to 1.8 mm, and the total attachment loss was up to 4.5 mm. That is, the chronic inflammatory process in the periodontium against the background of NAFLD was an important pathogenetic chain of comorbid patho­logy.

In the group consisting of spouses with NAFLD, only 9.0% had clinical manifestations of the second-degree CP, and 20.4% had the first-degree CP. In this group of studied patients, in contrast to their sick spouses, 13.6% had chronic catarrhal gingivitis (CCG), which inflammatory process covers only the gums without the involvement of other periodontal tissues, and the rest had no clinical manifestations of chronic inflammation in the periodontium. The OHI-S value did not significantly differ from the NAFLD data and demonstrated an unsatisfactory oral hygiene level. However, despite the more favorable objective data of healthy spouses, chronic inflammation in the periodontium was still present and was a source of systemic, pro-inflammatory cytokines demonstrated by an increase in the permissible CRP level.

In the control group, CCG was diagnosed in 4 (16.0%) persons and only 1 (4.0%) patient had localized CP provoked by traumatic occlusion.

Taking into account insulin resistance, overweight and low-intensity chronic inflammation, a negative feeding habit was determined — excessive consumption of sweetened beverages during the day, and a positive habit — compliance with twice-daily home oral hygiene, among spouses and compared with the control group. 77.2% of NAFLD patients had this feeding habit, especially such excessive consumption occurs during work (break/snack) on the subconscious level. According to questionnaire details, twice-daily oral hygiene was observed in only 56.8% of patients.

Among other family members not having NAFLD, the percentage of abusers of sweetened beverages was 54.5%, and those taking care of their oral cavity twice a day — 52.2%. Since in each married couple one of the members suffered from NAFLD in contrast to the other, the coincidence in habits between both spouses was analyzed. Half of the spouses (50.0%) equally consumed sweetened beverage in excess. At the same time, almost half of the spouses (47.7%) maintained oral hygiene twice a day. In 17 married couples, the spouses coincided as to the one-time oral hygiene care, usually in the morning. That is, this indicates a sufficiently high percentage of coincidence among married couples in a favorable or unfavorable direction, which is usually explained by the similarity in habits between both spouses.

But excessive consumption of sweets can also be a subconscious habit aimed at increasing the intake of glucose against the background of insulin resistance. This condition may contribute to changes in the sensitivity of tongue taste receptors to sweets, which may correlate with the HOMA-IR index.

According to threshold gustometry, none of the NAFLD patients and none of their spouses had sensitivity to the minimum dilution (0.5%) of glucose solution. Most NAFLD patients (47.7%) had the sensitivity of taste receptors to 2.5% glucose solution, 10 patients responded to 2.0% solution and 9 patients — to 1.5% solution. There were also 3 patients who felt only the maximum concentration — 5% glucose solution, and only 1 patient had sensitivity within normal limits — 1% glucose solution.

The reaction of taste receptors of more than half of healthy spouses (63.6%) was to 1.5% glucose solution. 13.6% of patients had sensitivity to 2% solution, and 2 persons — to 2.5% glucose solution. Among 18.2% of healthy spouses, the normal reaction to 1% glucose solution was recorded.

It should be noted that the control group indicators were taken as a norm of threshold gustometry, where the tongue taste receptors of 64% of persons reacted to 1% solution, and 16.0% — to 0.5% solution. Only 5 persons from the control group needed 1.5% glucose solution for sensation.

It is expected that the telomere length of gingival epithelial cells was the shortest in those NAFLD patients suffering from CP. It is evident that with the above-mentioned risk factors — endogenous (metabolic changes in case of NAFLD) and exogenous (habits) — systemic low-intensity, chronic inflammation causes a genotoxic effect reflected in the reduction of the length of telomeres of gingival epithelial cells. However, the sick the spouses also had a decrease in the length of telomeres of epithelial cells (Table 2).

Table 2. Data on threshold densitometry and length of the telomeres of gingival epithelial cells among the studied persons

The complex of treatment-and-prophylactic measures prescribed to patients allowed to improve both subjective and objective indicators. Thus, the NAFLD patients had a tendency to normalize metabolic indicators, while metabolic indicators of their spouses, in most cases, returned to the reference values. It is evident that the NAFLD treatment is a long process, but a certain improvement in the condition after 3 months demonstrates the correctness of treatment and prevention, namely, in the married couple.

When examining periodontal tissues, most patients in the main group also had a tendency towards clinical CP remission. However, the CP degree ratio changed, namely 1 patient had a rapid deterioration and CP transition from I to II degree. In general, the main group had a decrease in the chronic inflammation intensity, being a favorable change. The papillary bleeding index PBI indicated a gradual normalization of the periodontal microcirculatory bed. Despite the fact that the attachment loss did not change, however, the depth of periodontal pocket probing decreased by 0.3 mm due to the decrease in gingival edema confirming the less pronounced intensity of chronic inflammation in the periodontium. Unfortunately, the OHI-S index did not improve at all, but remained at the «unsatisfactory» level (Table 3).

Table 3. Data on the objective periodontal condition of the studied persons in the follow-up clinical observation

Those family members not suffering from NAFLD also had positive changes of the gum condition. Thus, although CP led to irreversible periodontal destruction, it was in the phase of clinical remission.

Repeated questionnaire survey regarding habits-exogenous risk factors for chronic inflammatory process in the periodontium demonstrated changes among both NAFLD patients and their spouses. On the part of dietary factors, a reliable improvement in the results was determined compared to the 1st visit. The number of NAFLD patients continuing to consume sweetened beverages decreased by almost 6 times. This feeding habit of healthy spouses also decreased by 12 times. The number of married couples where excessive consumption of sweets coincided between both spouses changed in a favorable direction: the number of married couples who adjusted their diet together increased by more than 5 times, and only 1 married couple turned out to be undisciplined (Table 4).

Table 4. Questionnaire data and other indicators among the studied persons in the follow-up observation

Changes in the sensitivity of tongue taste receptors to sweets also took place being a positive result of complex therapy of patients. The results of successful habit correction also demonstrated positive changes in the threshold gustometry data of healthy spouses.

Unfortunately, there was no significant increase in the number of persons in both the main group and the specified spouses adhering to the prescribed oral hygiene algorithm. The coincidence of spouses in the twice-daily oral hygienic care increased by only 4 pairs, and most family members coincided precisely in the single-time tooth brushing. Among the remaining married couples there was no coincidence — either the patient or his/her spouse brushed his/her teeth once in the morning. Perhaps the lack of motivation of patients to maintain a proper level of oral hygiene is related to the fact that the correction of feeding habit is double and occurs on the part of both the gastroenterologist and the dentist, while the correction of oral hygienic care is motivated only by the dentist.

However, due to the complex of treatment procedures, a decrease in the genotoxic effect took place, reflected in the improvement of the telomere length indicator being important prevention of premature aging of epithelial cells. The length of telomeres of gingival epithelial cells of patients was better compared to the 1st visit data, although it did not significantly reach the value of the control group of patients. Anyway, it was recorded that the healthy spouses had the value of the length of telomeres of epithelial cells at the control value level. Such data indicate the importance of correcting the negative impact of exogenous factors-habits of both patients and their spouses, since the length of telomeres is reduced due to physiological cell replication being a natural life cycle, and the barrier properties of the periodontal epithelial layer do not deteriorate.

Discussion

The CP is another source of supply of pro-inflammatory cytokines and toxins to the internal environment in NAFLD patients [8]. Such combined diseases mutually support the course, contribute to its deterioration and reduce the effectiveness of isolated treatment of pathogenetic components of comorbid pathology. The combination of NAFLD and CP acts as an endogenous factor based on metabolic disorders, but existing exogenous factors gradually lead to the formation of negative changes in metabolic processes [9]. The latter are understood to be the certain life habits, some of which will contribute to the state of «health», and others may be initiators of disorders.

Exogenous factors-habits contribute to the maintenance of the NAFLD course and the emergence of a source of chronic inflammation in the oral cavity — CP. In particular, the CP course is influenced by such an exogenous factor as compliance with twice-daily oral hygienic care [10, 11]. It is important for the patient not only to comply with twice-daily teeth brushing, but also to have the correct manual skills for home oral hygiene.

The analysis of home oral hygiene skills detected incorrect toothbrush movements, which does not certainly contribute to high-quality cleaning, even with twice-daily compliance. This was demonstrated by OHI-S in both NAFLD patients and their spouses in the «unsatisfactory» meaning. It is evident that such a level of home oral hygiene leads to the accumulation of food debris in the interdental spaces, in the cervical tooth area and will contribute to the opportunistic pathogenic microflora infectivity with the chronic inflammation initiation in the form of CCG, and subsequently CP. This is especially important for NAFLD patients, because CP is a component of comorbid pathology, and therefore the elimination of conditions for the development and course of chronic inflammation in the periodontium is considered a break in the «vicious circle» and an important component of the comprehensive treatment of metabolic disease [12–14]. In case of metabolic-associated diseases, conditions are created under which telomere shortening and, accordingly, premature cell aging are accelerated, while normal gradual shortening of telomeres of gingival epithelial cells, as a result of physiological replication, leads to aging and apoptosis, but there is no physiological dysfunctions of epithelial cells, because this is the course of a programmed life cycle [15].

However, the prevention of a healthy periodontal condition is relevant for spouses suffering from NAFLD, whose metabolic indicators are within normal limits, because pro-inflammatory cytokines and pathogenic microflora will pass from this source into the internal environment and the intestinal microbiome contributing to the initiation of chronic low-intensity inflammation and dysbiosis. Against the background of bad feeding habit (excessive consumption of sweets) and due to the risk of metabolic disorders in spouses suffering from NAFLD, the phenomena of hepatic steatosis are possible, and further stimulation of chronic inflammation on the part of CCG/CP can provoke steatohepatitis requiring drug treatment. Therefore, both the analysis and correction of bad habits should be carried out at the married couple level, which will better motivate both and contribute to the successful NAFLD patient treatment and the prevention of the initiation of metabolic disorders in his/her spouse.

Conclusion

Exogenous factors-habits contribute to the support of the NAFLD course and the emergence of a source of chronic inflammation in the oral cavity — CP.

Against the background of endogenous (NAFLD and CP) and exogenous (habits) risk factors, there is an acceleration of the telomere length reduction due to the changes with a genotoxic effect — systemic low-intensity, chronic inflammation, insulin resistance, etc.

The trend towards clinical CP remission is a favorable and important condition for the successful comprehensive comorbid pathology treatment, the monitoring of which should be carried out correctly at the married couple level, due to the coincidence of the usual lifestyle.

References

Надійшла до редакції/Received: 10.02.2025
Прийнято до друку/Accepted: 19.02.2025