INTRODUCTION:

Periodontal disorders are conditions related to the process of dental plaque formation. Chronic periodontitis is a multifactorial disease, in which there is destruction of the periodontal ligament, cementum and the alveolar bone leading to the apical migration of the junctional epithelium. Periodontal disease is initiated by bacterial colonisation of the tooth surface that triggers an inflammatory response in the adjacent host tissues.^1-4

Chronic periodontitis is caused by the Porphyromonas gingivalis (Pg) bacteria which accumulates in the gingival sulcus.^5,6 This bacteria causes the junctional epithelium to experience inflammation and loss of attachment to form a periodontal pocket.

The clinical sign of this disease is the depth of gingival sulcus that is more than 3mm. From the radiographic examination, the shape of alveolar bone resorption is horizontal.^7,8

In the junctional epithelium, there is a dominant adhesive protein that is laminin-5.^9,10,11 This protein plays an important role in the attachment of junctional epithelium to the tooth surface. ^12-17 Laminin-5 also has a role in cell adhesion and migration. This protein consists of 3 chains namely α3, β3, and γ2.¹⁸ Ccompared with healthy people, there is an increase in the concentration of laminin-5 chain γ2 (laminin-5γ2) in gingival crevicular fluid (GCF) peridontitis patients.¹⁹

Various attempts have been made to treat periodontitis, one of which is by applying medicinal plants. Medicinal plants have many advantages including affordable price, easy to obtain, and low side effects.²⁰ One of the medicinal plants in question is turmeric (Curcuma longa). The main active ingredient in turmeric is curcuminoid which contains curcumin as the main content.²¹

Curcumin (diferuloylmethane) is an orange-yellow pigment obtained from the rhizomes of Curcuma longa. Chemically, it is an extended pseudosymmetric polyphenol (diferuloylmethane). In recent years, in vitro and in vivo researchers have proposed that curcumin has anticarcinogenic, antioxidant, and anti-inflammatory effects besides antibacterial, antiviral, and antifungal properties and besides being a wound-healing accelerator. It is a potent inhibitor of transcription factors that play a role in inflammation, namely NFκB and AP-1.^22-24 Curcumin can reduce alveolar bone loss in rats with periodontitis through TNF-α and IL-6 inhibition.²⁵ Curcumin administered systemically can inhibit NFκB activity in gingival epithelial cells of rats with periodontitis.²⁶ 1% subgingival curcumin administration can reduce the level of gingival inflammation through the indicators of reduced bleeding on probing.²⁷

The anti-inflammatory properties of curcumin are mediated by modulation of the activity of signaling pathways and transcription factors, especially nuclear factorβ (NF-κβ), activating protein-1 and mitogen activated protein kinases (MAPKs). Down-regulation of the activation of and MAPKs by curcumin suppresses the expression of interleukin-6 (IL-6), IL-1β, tumor necrosis factor-α, matrix metalloproteinase 2 (MMP-2), and MMP-9 in NF-κβ in the late phase of experimental acute pancreatitis, in the modulation of arthritis, in the prevention and healing of indomethacin-induced gastric ulcer, and in the treatment of inflammatory bowel disease and Crohn’s disease. Curcumin is also reported to improve wound healing by increasing collagen deposition, angiogenesis, and the density of fibroblasts.

Interestingly, curcumin-treated wounds presented not only a greater number of fibroblasts but also more infiltrating macrophages and neutrophils compared with untreated wounds. The better regulation of granulation tissue formation by curcumin and induction of growth factors are key features, which can be extremely useful in the therapy of periodontal disease. This happens probably because of the activity of curcumin on AP-1 which plays a role in the synthesis of laminin-5γ2. In fact, curcumin is a potent inhibitor of NFκB and AP-1 transcription factor.²⁸

Based on this description, junctional epithelium migration occurs due to bacterial invasion causing inflammation such as periodontitis. Periodontitis causes loss of attachment which is an indicator of damage to the junctional epithelium. The purpose of this study is to observe the changes in the expression of protein laminin-5γ2 in junctional epithelium of 1% curcumin-treated rats with periodontitis.

MATERIALS AND METHODS:

The model of periodontitis rats used in this study was 5 months old male Rattus norvegicus with a bodyweight of 325-350 grams. The rats were divided into a control group (Pg) which consisted of 24 rats and a treatment group (Cur-Pg) which consisted of 24 rats. Each group was divided into 3-time observations (24 hours, 7th day, and 14th day) so that each group consisted of 8 periodontitis rats.

Periodontitis induction in this study was done using Pg bacteria (ATCC 33277). The bacteria were grown in media containing tryptic soy broth (TSB) and incubated in anaerobic conditions for 24 hours. Furthermore, the bacteria were cultured in blood agar containing hemin and vitamin K and were placed in an incubator anaerobically for 24 hours at 37⁰C. The greatest Pg colonies were moved into the liquid medium containing thioglycolate and then incubated for 24 hours at 37^oC in anaerobic environment. Then, with spectrophotometry of 624 nm wavelength, a bacterial concentration of 1x10⁶ CFU was made.

The gingival sulcus of the lower incisor teeth of the rats in the treatment group was exposed to 10⁶ CFU Pg bacteria in 30 μl (0.03 ml) PBS with micropipette every 3 days for 2 weeks.^21,22 At the time same, the gingival sulcus of the lower incisor teeth of the rats were irrigated with 1% curcumin two times a day as much as 30μl (0.03 ml) with a micropipette. While in the control group, there was no administration of curcumin.

The observations for the control and treatment groups were carried out for 24 hours on the 7th day and 14th day. The samples were obtained through the directional pieces of the lower incisor of the sagittal junctional epithelium. An examination of the samples was performed with immunohistochemistry to see the expression of laminin-5γ2. On the other hand, a statistical analysis was performed with the help of t-test (α = 0.05).

RESULTS:

In all groups, the normality test with Kolmogorov-Smirnov model for the expression of laminin-5γ2 showed a normal distribution of data (p>0.05). While on the other hand, a homogeneity test with the Levene test revealed that the expression of laminin-5γ2 between groups had an inhomogeneous variance. Moreover, the t-test between treatment groups (Pg and Cur-Pg) showed a significant difference that is by p< 0.05. The Brown-Forsythe test on inter-time groups (24hours, days-7, and days-14) also indicated significant differences (p<0.05), at least in between a pair of groups. Furthermore, a double comparison analysis was performed using the Games-Howell test to determine the differences in the expression of laminin-5γ2 for each treatment group at each time.

In the Pg group, the results of the Games-Howell test presented that the expression of laminin -5γ2 at 24 hours and at the 7th day of observation had no significant differences (p>0.05). Instead, a significantly different expression of laminin-5γ2 occurred in between the 24 hours, 7th day, and 14th day of observation (increased expression) (p<0.05). In Cur-Pg group, there also no significant difference that happened in within the 24 hours, 7th day, and 14th day of observation (p>0.05). However, in 24 hours observation and 14th day of observation, there was a significant difference. The laminin-5γ2 expression on Pg and Cur-Pg group on the first 24 hours and 7th day of observation did not show a significant difference (p>0.05) despite a decline in value. However, the observation on the 14th day illustrated that the expression of laminin-5γ2 between Pg and Cur-Pg group was different significantly (decreased expression) (p<0.05).

The results of measuring the expression of laminin-5γ2 on the junctional epithelium in the control group and the treatment group can be seen in Table 1 and Figure 1.

DISCUSSION:

Gingival attachment to the tooth surface is mediated by the junctional epithelium. The junctional epithelium is a front defense line of periodontal tissue. It is known that porphyromonas gingivalis (Pg) bacteria can damage the junctional epithelium causing loss of attachment and migration of junctional epithelium to the apical to form a pocket which then will result in periodontitis. Therefore, this study tries to describe the effort to inhibit the progression of periodontitis by administering 1% curcumin locally.

The invasion of Pg bacteria causes inflammation and damage to the gingival attachment to the tooth surface. The gingival attachment to the tooth surface is supported by an adhesive protein known as laminin-5. Laminin-5 protein consists of 3 chains such as α3, β3, and γ2. If the γ2 chain of the laminin-5 protein is fragmented in the short section of NH2 terminal due to proteolytic enzyme activity, this will result in cell migration.^29-32

This study seeks to inhibit the progression of junctional epithelium migration caused by Pg bacteria so as not to aggravate periodontitis. This effort is done by administering 1% curcumin locally.^33-36 Curcumin was used in this study because it has anti-inflammatory properties.^37-39 1% of curcumin concentration was also used as the basis of previous research. 1% curcumin was given to patients with periodontal disease by means of gargling by which it has been clinically proven to reduce the level of inflammation.^40-46

In other studies, 1% curcumin was administered orally to the rats and it was found that 1% curcumin can reduce the level of inflammation with laboratory indicators namely TNF-α, IL-6, and CP-1 levels in adipose tissue.⁴⁷

The result of this study are consistent with previous study by Krismariono, which claimed that local administration of 1% curcumin in the gingival sulcus can reduce the degree of inflammation in the periodontal tissues, especially the gingival junctional epithelium, by reducing the level of NFκB.⁴⁸

Yamamoto et al. Also claimed that flavonoid was able to suppress NFkB and Curcumin can suppress, TNF- α, prostaglandin level and IL-6.⁴⁹

The results of this study indicate that the local administration of 1% curcumin can reduce the expression of laminin-5γ2. This can be seen in the average laminin-5γ2 level in the Cur-Pg group which was lower than the Pg group. The differences began to be significant on the 14th day of observation. Whereas, in the first 24 hour and 7th day of observation, there were no significant differences even though the average level of laminin-5γ2 in Pg group was lower than the Cur-Pg group.

Curcumin inhibits the phosphorylation of IκB kinase so that the translocation of NFκB into the cell nucleus is inhibited which in turn will decrease the NFκB activity. In this case, AP-1 activity also decreases due to the inhibition from curcumin. The decreased activity of NFκB and AP-1 will have an effect on the decreasing of laminin-5γ2 synthesis because the synthesis of laminin-5γ2 is inseparable from the role of transcription factors for NFκB and AP-1. This mechanism might cause the expression of laminin-5γ2 in Cur-Pg group lower than in Pg group.

Based on the descriptions above, it appears that the exposure to Pg bacteria causes an increase in the expression of laminin-5γ2. The increased expression of laminin-5γ2 occurs especially in between the 7th day and 14th day. In the 1st day and 7th day, there was also an increase in the expression of laminin-5γ2 but it was not significant. Curcumin administration can inhibit the increase of laminin-5γ2 expression which can be seen in the 7th day and 14th day of observation. When compared between the Pg and Cur-Pg groups, the inhibition of the increased expression of laminin-5γ2 by curcumin was clearly seen on the 14th day of observation.

CONCLUSION:

The administration of 1% curcumin that is given locally on the gingival sulcus can reduce the expression of laminin-5γ2 in chronic periodontitis due to the existence of Porphyromonas gingivalis bacteria.

Table 1. The expression of laminin-5γ2 in the junctional epithelium at each group and time

Group	Time			t-test
Group	24 hours	7th day	14th day	t-test
Pg	13.02 + 1.516^a,b	19.83 + 5.359^a,b	27.81 + 2.959^c	p=0.001
Cur-Pg	12.07 + 1.129^a	15.41 + 3.126^a,b	16.58 + 2.192^b	p=0.001
Brown-Forsythe	p=0.000

Description: The same superscript shows no significant differences between groups (Games-Howell).

Figure 1. The expression of laminin-5γ2 in the junctional epithelium at each group and time

REFERENCES:

1. Camabala AD. Gajendran P. Role of Interleukin 1 in Gingival Tissues in Chronic Periodontitis -A Review. Research J. Pharm. and Tech. 2017; 10(9): 3185-3187. doi: 10.5958/0974-360X.2017.00566.2

2. Sanjeevi J. Gajendran P. Role of Interleukin 1 in serum in Chronic Periodontitis -A Review. Research J. Pharm. and Tech. 2017; 10(11): 4090-4092. doi: 10.5958/0974-360X.2017.00741.7

3. Inayathulla. Goudanavar P. Acharya A. Development and Evaluation of In-situ gel containing Linezolid and Diclofenac Sodium in the treatment of Periodontitis. Asian J. Pharm. Tech. 2020; 10(1):20-26. doi: 10.5958/2231-5713.2020.00005.7

4. Pavithra RS. Varghese S. Gingival Tissue Level of Interleukin-1 in Diabetic Patient with Chronic Periodontitis. Research J. Pharm. and Tech. 2017; 10(12): 4442-4444. doi: 10.5958/0974-360X.2017.00818.

5. Prithi R. Geetha RV. Static Effects of Fruits on Periodontitis. Research J. Pharm. and Tech. 2014; 7(3): 365-367.

6. Gayathri. M. Dr. Gheena. S. Gopinath. Comparison of the aerobic microbes in supragingival plaque of chronic and aggressive periodontitis patients. Research J. Pharm. and Tech. 2016; 9(9):1363-1366. doi: 10.5958/0974-360X.2016.00261.4

7. Newman MG. Takei H. Klokkevold PR. Carranza FA. Newman. Carranza’s clinical periodontology. 11th ed. Philadelphia London New-York. WB.Saunders Co. 2012; p: 17-31, 96-103, 54-164, 336-350.

8. Feng Z. Weinberg A. Role of bacteria in health and disease of periodontal tissues. Periodontology 2000. 2006; 40: 50-76

9. Thorup AK. Dabelsteen E. Schou S. Gil SG, Carter WG and Reibel J. Differential expression of integrins and laminin-5 in normal oral epithelia. APMIS. 1997; 105: 519-530. doi: 10.1111/j.1699-0463.1997.tb05049.x

10. Hormia M. Sahlberg CI. Airenne T. The epithelium-tooth interface - a basal lamina rich in laminin-5 and lacking other known laminin isoforms. J Dent Res. 1998; 77: 1479-1484. doi: 10.1177/00220345980770070201

11. Mullen LM. Richards DW. Quaranta V. Evidence that laminin-5 is a component of the tooth surface internal basal lamina supporting epithelial cell adhesion. J Periodontal Res. 1999; 34 (1):16-24. doi: 10.1111/j.1600-0765.1999.tb02217.x

12. Hormia M. Owaribe K. Virtanen I. The dento-epithelial junction: cell adhesion by type I hemidesmosomes in the absence of a true basal lamina. J Periodontol. 2001; 72:788-797. doi: 10.1902/jop.2001.72.6.788

13. Oksanen J. Sorokin LM. Virtanen I. Hormia M. The junctional epithelium around murine teeth differs from gingival epithelium in its basement membrane composition. J Dent Res. 2001; 80: 2093-2097. doi: 10.1177/00220345010800121401

14. Pollanen MT. Salonen JI. Uitto VJ. Structure and function of the tooth-epithelial interface in health and disease. Periodontology 2000. 2003; 31: 12-31. doi: 10.1034/j.1600-0757.2003.03102.x

15. Shimono M. Biological characteristics of the junctional epithelium. Journal of Electron Microscopy. 2003; 52 (6): 627-639. doi:10.1093/JMICRO/52.6.627.

16. Kinumatsu T. Hashimoto S. Muramatsu T. Sasaki H. Jung HS. Yamada S. Shimono M. Involvement of laminin and integrins in adhesion and migration of junctional epithelium cells. J Periodont Res. 2009; 44: 13-20. doi: 10.1111/j.1600-0765.2007.01036.x

17. Sugisawa M. Masaoka T. Enokiya Y. Muramatsu T. Hashimoto S. Yamada S. Shimono M. Expression and function of laminin and integrins on adhesion/migration of primary culture cells derived from rat oral epithelium. J Periodont Res. 2010; 45: 284-291. doi: 10.1111/j.1600-0765.2009.01231.x

18. Colognato H. Yurchencho PD. Form and function: the laminin family of heterotrimers. Developmental Dynamics. 2000. 218: 213-234. doi: 10.1002/(sici)1097-0177(200006)218:2<213::aid-dvdy1>3.0.co;2-r

19. Emingil G. Kuula H. Pirila E. Atilla G. Sorsa T. Gingival crevicular fluid laminin-5 γ2-chain levels in periodontal disease J Clin Periodontol. 2006; 33: 462-468. doi: 10.1111/j.1600-051x.2006.00933.x

20. WHO. Traditional medicine strategy 2002-2005. 2002; 1-3. https://apps.who.int/iris/handle/10665/67163

21. Aggarwal BB. Bhatt ID. Ichikawa H. Ahn KS. Sethi G. Sandur SK. Natarajan C. Seeram N. Shishodia S. Curcumin: biological and medicinal properties. 2006; p: 297-340

22. Chattopadhyay I. Biswas K. Bandyopadhyay U. Banerjee RK. Turmeric and curcumin: biological actions and medicinal applications. Current Science. 2004; 87 (1): 44-49

23. Jurenka JS. Anti-inflammatory properties of curcumin. a major constituent of curcuma longa. A review of preclinical and clinical research. Alternative Medicine Review. 2009; 14 (2): 141-153

24. Sha AM. Garib BT. Antibacterial Effect of Curcumin against Clinically Isolated Porphyromonas gingivalis and Connective Tissue Reactions to Curcumin Gel in the Subcutaneous Tissue of Rats. Research Article of BioMed Research International. 2019; (2019) doi.org/10.1155/2019/6810936

25. Zhou T. Chen D. Li Q. Sun X. Song Y. Wang C. Curcumin inhibits inflammatory response and bone loss during experimental periodontitis in rats. Acta Odontol Scand. 2013; 71(2): 349-56. doi: 10.3109/00016357.2012.682092

26. Guimaraes MR. Coimbra LS. de Aquino SG. Spolidorio LC. Kirkwood KL. Rossa C. Potent anti-inflammatory effects of systemically administered curcumin modulate periodontal disease in vivo. J Periodont Res. 2011; 46: 269-279. doi: 10.1111/j.1600-0765.2010.01342.x

27. Suhag A. Dixit J. Dhan P. Role of curcumin as a subgingival irrigant: A pilot study. Qiuntessence Journals. 2007; 4 (2):115-121

28. Sreedhar A. Sarkar I. Rajan P. Pai J. Malagi S. Kamath V. Barmappa R. Comparative evaluation of the efficacy of curcumin gel with and without photo activation as an adjunct to scaling and root planing in the treatment of chronic periodontitis: A split mouth clinical and microbiological study. J of Natural Science, Biology and Medicine. 2015; 6: 102-109. doi: 10.4103/0976-9668.166100

29. Suzumura Y. Kameyama Y. Mizutant M. Kato M. Kondo K. Mabuchi R. Long junctional epithelium produced by application of bacterial protease in rats. J Periodont Res. 1989; 24: 217-221. doi: 10.1111/j.1600-0765.1989.tb02009.x

30. Polak D. Wilensky A. Shapira L. Halabi A. Goldstein D. Weiss EI and Haddad YH. 2009. Mouse model of experimental periodontitis induced by Porphyromonas gingivalis / Fusobacteriumnucleatum infection: bone loss and host response. J Clin Periodontol. 2009; 36: 406-410. doi: 10.1111/j.1600-051x.2009.01393.x

31. Giannelli G. Falk-Marzillier J. Schiraldi O. Stetler-Stevenson WG. Quaranta V. Induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5. Science. 1997; 277: 225-226

32. Gagnoux PL. Allergra M. Spirito F. Pommeret O. Romero C. Ortonne JP. Meneguzzi G. The short arm of the laminin g2-chain plays a pivotal role in the incorporation of laminin 5 into the extracellular matrix and in cell adhesion. Journal of Cell Biology. 2001; 153: 835-849. doi: 10.1083/jcb.153.4.835

33. Chen D. Nie M. Fan MW. Bian Z. Anti-inflammatory activity of curcumin in macrophages stimulated by lipopolysaccharides from Porphyromonas gingivalis. Pharmacology. 2008; 82 (4): 264-9. doi: 10.1159/000161127

34. Kuo JJ. Chang HH. Tsai TH. Lee TY. Positive effect of curcumin on inflammation and mitochondrial dysfunction in obese mice with liver steatosis International Journal of Molecular Medicine. 2012; (30): 673-679. doi: 10.3892/ijmm.2012.1049

35. Philip S. Kundu GC. Osteopontin induces nuclear factor kappa B-mediated promatrix metalloproteinase-2 activation through IkB/IKK signaling pathways and curcumin (diferulolylmethane) down-regulates these pathways. J Biol Chem. 2003; 278:14487-14497. doi: 10.1074/jbc.m207309200

36. Verma UP. Abhaya G. Disha S. Role of Curcuma longa in the Management of Gingivitis. International J of Public Health Science (IJPHS). 2018; 7 (3). 216-222. doi:10.11591/ijphs.v7i3.14830

37. Mohammad CA. Efficacy of Curcumin Gel on Zinc, Magnesium, Copper, IL-1β, and TNF-α in Chronic Periodontitis Patients. Research Article. BioMed Research International. 2020; doi.org/10.1155/2020/8850926

38. Livada R. Shiloah J. Tipton DA. Dabbous MK. The Potential Role of Curcumin in Periodontal Therapy: A Review of the Literature. J of Int Aca Periodon. 2017; 19(3):70-79.

39. Anusha D. Chaly PE, Junaid M. Nijesh JE. Shivashankar K. Sivasamy S. Efficacy of A Mouthwash Containing Essential Oils and Curcumin As An Adjunct To Nonsurgical Periodontal Therapy Among Rheumatoid Arthritis Patients With Chronic Periodontitis: A Randomized Controlled Trial. Original Research. Indian J of Dental Research. 2019; 30 (4). 506-511. doi: 10.4103/ijdr.ijdr_662_17

40. Kaur S. Sharma R. Sarangal V. Kaur N. Prashar P. Evaluation of Anti-Inflammatory Effects of Systemically Administered Curcumin, Lycopene and Piperine As An Adjunct To Scaling And Root Planing: A Clinical Study. Original Article. An. International Quarterly J Of Research in Ayurveda. 2017; 38 (2). 117-121. doi: 10.4103/ayu.ayu_63_17

41. Lubis P. Nasution R Ilyas S, Ervina I, Primasari A. Effectiveness of 1% curcumin gel subgingival application on interleukin-6 levels in chronic periodontitis patients. International Journal of Applied Dental Sciences. 2020; 6(3): 513-516

42. Susanto A. Amaliya A. SWN C. The Effect of 2% White Turmeric (Curcuma zedoaria) Gel as Adjunctive Therapy of Scaling and Root Planing Treatment toward the Interleukin 6 Level in Chronic Periodontitis. Int J of Chem Res. 2018; 11(08):182-87. doi.org/10.20902/ijctr.2018.1108

43. Reddy SV. Suresh J. Hemant KS. Yadav. Singh A. A Review on Curcuma longa. Research J. Pharm. and Tech. 2012; 5(2):158-165.

44. Prafulla S. Lata P. Priya R.Vidya S. Novel Curcumin Derivatives: Targeted for Anti-Inflammatory Activity. Asian J. Research Chem. 2019; 12(2):49-54. doi: 10.5958/0974-4150.2019.00011.7

45. Das A. Roy A. Rajeshkumar S. Lakshmi T. Anti-inflammatory Activity of Turmeric Oil Mediated Silver Nanoparticles. Research J. Pharm. and Tech. 2019; 12(7):3507-3510. doi: 10.5958/0974-360X.2019.00596.1

46. Ritmaleni. Sardjiman. Purwantini I. Antimicrobial Activity of Curcumin Analog PGV-6, HGV-6 and GVT-6. Research J. Pharm. and Tech. 2021; 14(2):599-604. doi: 10.5958/0974-360X.2021.00107.4.

47. Xiao J. Yu XJ. Xie JL. Liu S. Li S. Protective effect and related mechanisms of curcumin in rat experimental periodontitis. Research. Head & Face Medicine. 2018; 14:12 doi.org/10.1186/s13005-018-0169-1

48. Krismariono A. The Decreasing of Nfκβ Level in Gingival Junctional Epithelium of Rat Exposed to Porphyromonas gingivalis With Application Of 1% Curcumin on Gingival Sulcus. Research Report. Dental J. 2015; 48(1): 35–38. doi.org/10.20473/j.djmkg.v48.i1.p35-38

49. Yamamoto Y. Gaynor RB. Therapeutic Potential of Inhibition of the NF-κβ Pathway in The Treatment of Inflammation and Cancer. J Clin Invest. 2001; 107 (2):135-142. doi: 10.1172/jci11914

Received on 28.04.2021 Modified on 06.02.2022

Research J. Pharm. and Tech. 2022; 15(7):3096-3100.

DOI: 10.52711/0974-360X.2022.00518