Curcumin Effect on Nitropropane Alterations on the
IL-1β, Tnfα & IL 6 in Submandibular Gland of Mice
Badawy M Abd El Hamied1* and Atef I Ahmed2
1Department of Oral Biology, Assiut University, Egypt
2Department of Oral Biology, Al-Azhar University, Egypt
Submission: April 08, 2019; Published: May 16, 2019
*Corresponding author: Badawy M Abd El Hamied, Lecturer, Department of Oral Biology, Faculty of Dentistry, Assiut University, Egypt
How to cite this article: Badawy M Abd El Hamied, Atef I Ahmed. Curcumin Effect on Nitropropane Alterations on the IL-1β, Tnfα & IL 6 in Submandibular
Gland of Mice. Adv Dent & Oral Health. 2019; 10(5): 555800. DOI: 10.19080/ADOH.2019.10.555800
Objectives: 2-Nitropropane (2-NP) is a rat liver carcinogen. Curcumin has shown some therapeutic activities. This study aimed to investigate if curcumin has a synergistic effect on the 2-NP alterations on the IL-1β, IL 6 & TNFα in submandibular gland of Mice.
Methods and materials: The gene expression levels of TNF-α, IL-1β & IL-6 were tested in submandibular gland of mice by RT-qPCR. 30 adult BALB/c male mice were divided randomly into 3 equal groups. Group I, a control group (n=10), injected with vehicle alone (canola oil, 5mL/kg). The latter two groups (n=20) by intraperitoneal injection of 2-NP (100mg/kg body weight dissolved in canola oil) 2 time/week for 4 weeks. At the same time of 2-NP injection, mice of group III were orally drinking water supplemented with curcumin 50(Mg/Ml)/4weeks. Submandibular gland tissues were histologically studied.
Results: the gene expression analysis showed a statistically significant upregulation of TNF-α, IL-1β, and IL-6 in group II. While, group III showed significantly downregulated expression. The apparent histopathological changes manifested in group II was less in group III.
Conclusions: Curcumin may modulate the alterations of 2-NP by down-regulating proinflammatory cytokines IL-1β, IL6 and TNF α in BALB/c mice.
The submandibular glands are major salivary glands located beneath the floor of the mouth. Interleukins are a group of cytokines that expressed by leukocytes . IL1 β is participating in the regulation of immune responses, inflammatory reactions, and hematopoiesis . IL-6 plays a role in the final differentiation of B cells into immunoglobulin-secreting cells . Several genetic modifications or mutations associated with dysregulated IL-1 activity and autoinflammatory disorders were identified . Expression of IL-1 is up regulated in different tumor phenotypes and is implicated as an important factor in tumor progression via expression of metastatic, angiogenic genes and growth factors. Therefore, down regulation of expression of IL-1 may be able to inhibit cancer progression . The miR146a and miR147b were associated with increased expression of genes related to the immune/inflammatory response. Overexpression of miR147b reduced the expression of the pro-inflammatory mediators IL-6 after IL-1β stimulation .
Nitropropane (2-NP) is a colorless, oily liquid with a mild odor. It is used as a solvent, principally in blends, and has many
industrial applications . Previously, severe liver damage, as
well as some kidney damage, has been observed in workers fatally poisoned from acute inhalation exposure to high concentrations of 2-nitropropane . The results indicated that 2-NP inflicted DNA damage in the bone marrow cells and thus could be leukemogenic . Studies were carried out in the rat indicated that 2-NP induced chromosome aberrations as well as DNA repair in vivo .
Curcumin and its two related compounds are the main secondary metabolites of Curcuma longa . Curcuma longa has been used in herbal medicines for the treatment of a variety of ailments such as rheumatism, diabetic ulcers, anorexia, cough and sinusitis. It has also been shown to have significant wound healing properties .
It has been reported to have anti-inflammatory, antioxidant, anti-rheumatoid, and anti-atherosclerosis role. It can also reduce lipid, eliminate free radicals and inhibit the growth of the tumor. Many reports had suggested that curcumin has shown great potential in the treatment of tumors by inducing apoptosis. Use of curcumin mouthwash has shown antiplaque and antigingivitic properties. It could be effectively used as an adjunct to scale and root planning . Also, it showed that ultra-soluble curcumin could prove useful as a therapeutic intervention in Sjögren’s
syndrome . Also, it inhibited IκB kinase β kinase activity in the
saliva of head and neck squamous cell carcinoma cancer, and this
inhibition correlated with reduced expression of several cytokines
The aim of this study was to investigate if curcumin has a
synergistic effect on the nitropropane alterations and the role of
IL-1β, IL 6 and TNFα.
All experimental animals maintained and monitored in a
specific pathogen-free environment. All experimental animal
protocols were performed according to regulations set by the
Institutional Animal Care and Use Committee and were approved
by ethical committee of faculty of veterinary medicine, Assiut
University (approved by faculty council number 4, at 25/4/2018).
All animal procedures were also performed according to the
Declaration of Helsinki and the guidelines for the care and use
of experimental animals established by the National Institutes of
Health (NIH). All animals could acclimatize in plastic cages (five
animals per cage) inside a well-ventilated room for one week prior
to the experiment. The animals were maintained under standard
laboratory conditions (temperature of 23 °C, relative humidity
of 60-70%, and a 12h light/dark cycle) and were fed a diet of
standard commercial pellets and water containing libitum. We
made every effort to minimize animal stress.
After 1 week of acclimatization, mice were randomly
categorized into three main groups (10 mice each).
a. Group I (control mice).
b. Group II (2- Nitropropane inject group mice).
c. Group III (2- Nitropropane inject group treated with
Oral toxicity was induced in mice in the latter two groups (n =
20) by intraperitoneal (i.p.) injection of 2- Nitropropane (100mg/
kg body weight dissolved in canola oil) 2 time/week for 4 weeks;
mice in the control group were injected with vehicle alone (canola
oil, 5mL/kg). At the same time of 2- NP injection, mice were orally
drinking water supplemented with curcumin 50 (Mg/Ml)/ 4
All animals were sacrificed at day 31 post- 2- NP injection.
The submandibular salivary gland was removed and cut into small
pieces in sterile saline. The pieces were suspended in Trizol for
RNA extraction and gene expression analysis.
The salivary gland pieces fixed in 10% formalin were microtechnically
processed in order to obtain paraffinized tissue
sections. The tissue sections were stained with hematoxylin and
eosin (H&E) for morphological examination and viewed under
The effect of curcumin administration of both on the expression
of a panel of selected genes involved in the inflammation and
toxicity of oral mucosa were investigated using real-time
After performing the indicated treatments, RNA was extracted
and then the corresponding cDNA was prepared, and then real
time PCR was applied for gene expression analysis as previously
described . In brief, all cDNA samples were processed in a 96-
well plate using the following cycling conditions: 10 minutes at
95℃, and 40 cycles at 95℃ for 15 seconds ended by one min. at
60℃. These data were analyzed according to Livak et al. .
Microscopically, the submandibular glands of BALB/c male
mice of the control group appeared with a well-known histologic
feature with no observable lesions. Mice in 2- Nitropropane
treated group appeared with marked atrophy and degeneration
of the acinar cells and ducts (Figure 1A). Glands in this group
showed also cytoplasmic vacuolations (Figure 1B). Ductal inflammatory cell foci in the submandibular glands were apparent
in this group (Figure 1C). In animals treated with curcumin there
was apparent improvement from the 2-NP deleterious effect. The
acinar architecture of the gland appeared near normal (Figure
1D). However, this reversal of 2-NP effect did not acquire the full
normal case of the gland.
Real time- PCR and mouse primers specific for IL-1β, IL-6
and TGFα were used to detect the presence of mRNAs. IL-1, IL-6
and TGFα could be detected in mouse submandibular gland in all
groups. The data shown in Table 1 indicate a dramatic difference
between different study groups. The means of IL-1β, IL-6 and
TGFα levels demonstrated a significant increase in mice with
2-NP when compared to controls (p<0.001). In contrast, use of
curcumin revealed that the relative mRNA levels of the IL-1β, IL-6
and TGFα decreased significantly compared with the 2-NP treated
mice (Figures 2-4).
Figure 2 shows that expression of mRNA for IL-1β genes
steadily increased with the administration of 2-NP (P < 0.001).
Significantly lower levels were observed with curcumin treatment
P< 0.05. As shown in Figure 4, the expression of IL-6 in tissue
samples was significantly lower after curcumin. The differences
in mean IL-6 expression appeared statistically significant, with
a P<0.01. However, tissue expressions of IL-6 in 2-NP treated
group had risen. As shown in Figure 4 expressions of TNFα were
significantly lower (P< 0.05) after curcumin administration.
Nowadays there is great evidence of the possible health
benefits of curcumin, but little is known about the effects on the
salivary glands. In the present work it was investigated that the
expression levels of IL-1β, IL-6 and TNF-α in the submandibular
glands of BALB/c mice were significantly higher in the 2-NP treated
animals compared to the control group. The histopathologic
analysis stated that the acinar cells were atrophic and degenerated.
In addition, these cells showed cytoplasmic vacuolations and
ductal inflammatory cell foci indicating focal inflammation.
These changes were comparable to those demonstrated in other
studies [18,19]. It was suggested that the liver damage induced
by 2-NP is related to oxidative damage, lipid peroxidation .
Previous results indicated that 2-NP inflicted DNA damage in the
bone marrow cells  and induced chromosome aberrations as
well as DNA repair . In contrast, the findings of this study
suggested that the expression levels of IL-1 IL-6 and TNF-α in
the submandibular glands from mice treated with curcumin
were significantly decreased. Histologically, there was partial
improvement from the 2-NP deleterious effect.
These results may be correlated with several studies which
evidenced possible health benefits of curcumin. A few studies had
shown that curcumin can be prevention and a chemotherapeutic
agent for colon, skin, oral and intestinal cancers. Curcumin was
also well known for its ant inflammatory and antioxidant, anticoagulant
and anti-infective effects [12,21,22]. The anticancer
effect of curcumin is attributed to activation of apoptotic
pathways in cancer cells, as well as inhibition of inflammation and
angiogenesis in the tumor microenvironment and suppression
of tumor metastasis. Several reports had demonstrated that
curcumin inhibits angiogenesis in a wide variety of tumor cells
through the modulation of various cell signaling pathways which
involve transcription factors, protein kinases, growth factors
and enzymes . Oral administration of curcumin beneficially
modulates many diseases including diabetes, fatty liver disease,
atherosclerosis, arthritis, cancer and neurological disorders .
Curcumin given 24 hours before irradiation reduced the structural
damage to the salivary glands .
In respect to the role of cytokines, expression of IL-1 is up
regulated in different tumor phenotypes and is implicated as an
important factor in tumor progression via expression of metastatic,
angiogenic genes and growth factors. Therefore, down regulation
of expression of IL-1 may be able to inhibit cancer progression .
Similarly, Interleukin 6 (IL6) and TNF α are involved in a wide
variety of biological functions. Tumor necrosis factor receptor-1
(TNFR1) is involved in apoptosis through extrinsic pathway
initiation. The level of soluble TNFR1 is reported increased in
primary Sjögren’s syndrome patients .
Furthermore, it was found that stimulation with IL-1β and
TNF-α increased submucosal gland secretion in a concentrationdependent
manner. The cytokine effect was dependent on cAMP.
Most importantly, IL-1β- and TNF-α-stimulated secretion was
blocked by the cystic fibrosis transmembrane conductance
regulator (CFTR) blocker. Also, during bacterial infections and
resulting release of proinflammatory cytokines, the glands are
stimulated to secrete fluid, and this response is mediated by
cAMP activated CFTR, a process that would fail in patients with CF
. It was demonstrated that IL6 pretreatment prevented both senescence and salivary gland hypofunction via a mechanism
involving enhanced DNA damage repair. It was indicated that
cellular senescence is a fundamental mechanism driving radiationinduced
damage in the salivary gland and suggested that IL6
pretreatment may represent a promising therapeutic strategy to
preserve salivary gland function in head and neck cancer patients
undergoing radiotherapy .
Additionally, higher expression of IL-6 was found in salivary
gland cancer. There was a high association of cytomegalovirus
CMV antigen presence with the presence of IL-6, and with the IL-6
expression intensity. Positive expression of CMV antigens in a high
percentage of SGC cells suggested that it might play an important
role in carcinogenesis by increasing IL-6 production and leading
to inhibition of apoptosis and tumor development .
Likewise, protein expression levels of IL-17 and IL-6 were
detected parotids and submandibular glands by ELISA. Compared
with the normal group, mRNA transcriptional levels and protein
expression levels of IL-17 and IL-6 were significantly up-regulated
after administration of deionized water .
In This study there was a strong association between administration
of curcumin and the expression levels of IL-1β, IL-6 and
TNF-α which can be interpreted by many authors. Elevated expression
and protein level of IL-1α and IL-1β were found in lymphoma
bearing mice, which were significantly down regulated by
curcumin treatment. Similarly, curcumin treatment down regulated
activation of IL-1α and IL-1β via AP-1 and NF-IL-6 respectively
(5). It has been shown to interfere with multiple cell signaling
pathways, including cell cycle, apoptosis, proliferation, survival,
invasion, angiogenesis, metastasis and inflammation .
Finally, the findings of this study conclude that curcumin
may attenuate the alterations of 2-NP by down-regulating
proinflammatory cytokines IL-1β, IL6 and TNF α in BALB/c mice.