The Effect of Curcumin on the Newborn Bone Development in Pregnant Rats that are Given Nicotine

Seher Yilmaz1*,Ayşe Yeşim Göçmen2, Adem Tokpinar1, Halil Yilmaz3, Mehtap Nisari4, Tolga Ertekin5, Şükrü Ateş1, Erdoğan Unur4 and Seda Avnioğlu6 1Department of Anatomy, YozgatBozok University Faculty of Medicine, Turkey 2Department of Biochemistry, YozgatBozok University Faculty of Medicine, Turkey 3Department of Therapy and Rehabilitation, Kozakli Vocational School, NevsehirHaciBektasVeli University, Turkey 4Department of Anatomy, Erciyes University Faculty of Medicine, Turkey 5Department of Anatomy, Afyonkarahisar Health Sciences University, Faculty of Medicine, Turkey 6Alanya AlaaddinKeykubat University, Faculty of Medicine, Department of Anatomy, Turkey


Introduction
Smoking is now an important public health problem in all ountries and is one of the reasons for preventable deaths in many countries. Around 7 million people die due to smoking every year in the world [1]. The increase in female users causes more problems especially during pregnancy [2,3]. Smoking during pregnancy can cause low birth weight, respiratory diseases and cancer sensitivity in infants [4]. Nicotine is the most important toxic component of cigarette in offsprıngs. That inhibits the activity of osteoblasts increases osteoclast differentiation from osteocytes and reducing the mechanical strength of bone. Low birth weight and skeletal development deformations in rat pups due to nicotine use during pregnancy and lactation have been reported [5,6]. Various agents are used to prevent these teratogenic effects of Nicotine. The most frequently studied agents are natural antioxidants [7]. Previous studies have shown that curcumin is a powerful antioxidant and is used in some cancer studies because of its antioxidant properties and also supports bone development [8][9][10]. Today, various experimental studies are carried out to determine the effects of harmful substances on bone development; and one of these studies is the Double Skeleton Staining Method. The process of staining bone and cartilage structures with hdyes is called Double Skeleton Staining [7].
Studies in which double skeletal staining has been used are grouped into two as Teratogenic Studies and Developmental Studies.
Teratogenic effects, skeletal system effects and morphological studies on fetuses have been carried out. The dual skeletal staining method on the skeletal system is frequently used [7,11]. Oxidative stress is a principal parameter for evaluating nicotine effect on rat bone [12]. Nicotine at high doses to cause miralizaton loss and loss of bone mechanical strength in intact rats. The results of the previous studies showed that nicotine is a risk factor for osteoporosis [13,14]. According to previous studies, curcumin has been shown to have inhibitory and delaying effects on the osteoporosis process.
Studies have shown that patients with significant bone injury after 6 months in curcumin-treated groups have decreased osteoporosis progression and bone turnover markers [15]. Nicotine is a harmful chemical which injures bone at development stage. In this study hypothesized that curcumin could play critical role on normalizing oxidative and inflammatory parameters which upregulated by nicotine. Therefore, we aimed to evaluate the effect of Low Dose Curcumin (LDC) and High Dose Curcumin (HDC) on humerus bone tissue after investigated with nicotine.

Material and Methods
The present study was conducted in accordance with the During the experiment rats were kept at a constant temperature of 19 -21ºC and in a 12 hour light / dark environment. Experimental groups were formed through dividing pregnant rats into 6 groups.
Invasive processes (ı.p,s.c) were applied to the rats for 20 days during the pregnancy; and the gestational period lasted 21 days in average (Table 1).

Dissolution and Sterilization of Curcumin and Nicotine
Nicotine and Curcumin were obtained from Sigma-Aldrich.
PBS (Phosphate Buffer Saline) was used as a solvent for nicotine.
Curcumin was dissolved in different volumes to provide the desired concentrations for each experimental group with DMSO (dimethyl sulfoxide) and PBS buffer. The DMSO ratio was adjusted to 0.05% of the total solution, the resulting solution was diluted with PBS; and the solutions prepared daily were sterilized by filtration.

Manipulation of Offspring
The offspring which were born were taken under Ketamine (75 mg / kg) + Xylazine (10 mg / kg) anesthesia. After cleaning the abdomen regions of the offspring with 70% alcohol, the abdominal walls were removed with a transverse incision, internal organs were removed, and the offspring were subjected to the Double Skeletal Staining Method (Table 2). For morphometric measurements, the images of the offspring were taken with Nikon E5700 camera via a stereomicroscope and were transferred to the computer. The length and area measurements of the bones in the images were measured using the Image J Program.

Inflammation Parameters and Ca +2
Protein samples were thawed, and commercial ELISA kits was calculated according to [18].

Statistical Analysis
All analyses were conducted using SPSS version 23.0 (IBM Co., NY, USA). Data were presented as the mean ± standard deviation.
For analysis of the differences in continuous variables among the groups, data were analyzed using one-way analysis of variance (ANOVA) followed by the post hoc Tukey's test for parametric data and the Kruskal -Wallis test followed by the post hoc Dunn's test for nonparametric data. Statistical significance was defined as a two-tailed P < 0.05.

Effects on Upper Extremity Long Bones
In study, long bones (humerus, radius, ulna) of the upper extremities were evaluated. Generally, when 6 mg/kg nicotine was given, there was a statistically significant decrease in the region lengths and ossification percentages, which showed the ossification in these bones (P < 0.05) When the additional curcumin was given, it was determined that the ossification increased and approached to that of the Control Group (Figure 1), (Table 3).

Effects on Lower Extremity Long Bones
In study, long bones of the lower extremity (femur, tibia and fibula) were also evaluated. A statistically significant decrease was found in the percentage of ossification in only the nicotinetreated group (P < 0.05). It was determined that ossification was more likely to reach the values of the Control Group in additional curcumin to nicotine and curcumin groups (Figure 2), (Table 4).
In our study, the average offspring size was lower in the groups to which nicotine was administered compared to the control group and to the group to which curcumin was administered. There were dead births in the groups to which nicotine was administered; and it was determined that maternal food consumption decreased in the nicotine-administered group (Table 5).

Discussion
Nicotine easily passes through the placenta due to its lipophilic nature. Several researchers used different doses of nicotine in rats during pregnancy in their experimental studies. In the literature, these doses range from 1.67 mg / kg to 7.5 mg / kg. [7,19,20].
investigated the protective effects of melatonin against the effects of nicotine on bones in their study; and found that the percentage and rate of ossification was low in the upper and lower extremity bones of the group to which 6 mg/kg nicotine was administered (31.42 ± 10.33) compared to the control group (43.71 ± 2.33). In their study, they stated that this ratio was close to the Control Group in the melatonin group [7,21]. investigated the negative effects of nicotine on bone development. In their study, they administered 3 mg/kg nicotine given i.p. during pregnancy and lactation. At the end of the experiment, it was reported that femur lengths of 21 days old offspring in the group treated with nicotine (19.1 ± 1.6 mm) were significantly lower than the control group (21.1 ± 0.3) and in their study they shown maternal nicotine exposure resulted in decreased birth weight, pregnancy weight gain, and bone lengthening, and increased apoptosis [21]. The lactation period of the mothers who smoked was measured for 24 hours in the study of [22], which were evaluated as 113 ± 179 ml in the Control Group and 47 ± 122 ml in the group in which mothers smoked [22,23].injected 2 mg/kg nicotine s.c into pregnant rats in their studies; and determined the dose of 2 mg/kg nicotine as 12 cigarettes per day. In their study, they reported that total cholesterol values in mother serum were higher than controls [23,24]. studied the teratogenic effect of nicotine in female fetuses exposed to cigarette smoke for 14 days before pregnancy and for 20 days during pregnancy, and male rats that were exposed to cigarette smoke for 28 days before pregnancy. In rats exposed to 18.6 ± 2.07 mg / m 3 Nicotine and 600 mg, 41.8 ± 3.71 mg / m 3 , a significant increase was reported in the number of missing or non-ossified bones in rats exposed to nicotine [24,25] evaluated the osteoclast genesis and endochondral ossification of long bones of fetuses in pregnant rats exposed to nicotine. In their study, they used 2 mg/kg nicotine s.c for 20 days in pregnant rats.
As a result of their experiments, they reported a significant decrease in the length of femur in fetuses exposed to nicotine compared to control group [25]. There are many studies conducted on pregnant rats and individuals showing that nicotine causes a decrease in fetus ossification rate. In the literature, this effect has been reported to be reduced by means of various antioxidant agents [3]. One of these substances is curcumin. Curcumin is used as a colorant in spices, food and textiles, and in diets, as well as in many diseases [26]. Many different pharmacological activities and biological benefits of Curcumin have attracted considerable attention in recent years [27]. Many studies have shown that Curcumin has anti-oxidant, anti-carcinogen, anti-inflammatory, anti-allergic, anti-dementia effects and is a free radical scavenger [28][29][30]. gave 30 mg / kg curcumin to the rats treated with periodontitis and reported that alveolar ossification increased [28,29].investigated the effect of curcumin on glucocorticoid-induced osteoporosis in rats and reported that curcumin increased osteoblast activity and decreased osteoclast activity [29,30] [32]. When bone fractures occur, a remarkably high yield of radicals is generated. It is suggested that as a break occurs, the minimal crystallites separate at grain boundaries with no major chemical changes, but the tightly bound collagen strands running through the mineral phase are forced to break homiletically. Some react with oxygen and yield oxygen radical metabolites [33].
Cigarette smoke is a complex mixture of more than 4700 chemical compounds including free radicals and oxidants. Toxicity exhibited by cigarette smoke may be due to combined action of these compounds inducing many cellular processes mediated through Reactive Oxygen Species (ROS). Major player probably nicotine as it is present in tobacco, in higher concentrations. Meanwhile, elevated levels of ROS can damage proteins, lipids, and DNA, eventually trigger oxidative stress and leading to cell death [34,35]. Oxidative damage to bio-macromolecule has been proved in the etiology of a wide variety of acute and chronic diseases, including osteoporosis [36].

Measurement of TOS, TAS, Vitamin D and SOD is a crucial
biomarker to evaluate oxidative damage [37][38][39][40][41]. In this study we detected that curcumin increased the TAS level whereas decreased the TOS levels both nicotine threated groups and nicotine untreated groups. The highest value of SOD was observed in Nicotine group and as well as the lowest value was recorded at control group.
Nicotine significantly increased SOD level by stimulating reactive oxygen system thus as an antioxidant curcumin slightly decrease the effect of nicotine and approximate the values to control group.
Vitamin D is an antioxidant ant it decreases when cell damaged [42]. In Nicotine group Vitamin D has the lowest value and Control group has the highest value. After nicotine treatment due to the ROS activity Vitamin D level significantly decreased but after dose dependent curcumin treatment it increased slightly.

GSH, present in concentrations of 2-10 mM within cells, is
the primary determinant of the cellular redox environment and exists mainly as the biologically active reduced-thiol form [43].
The oxidation of GSH to GSSG and subsequent decrease in the GSH/GSSG ratio is often associated with oxidative stress. Thus, the GSH/GSSG ratio is a simple and useful indicator of cellular redox state [44,45]. In the current study GSH decreased by the effect of The serum concentrations of those markers increase after nicotine treatment [46,47]. In the current study IL-6, IL-1β and TNF-α levels in Nicotine group were markedly increased in rats. Previous studies showed that curcumin treatment act as anti-inflammatory agent [48,49]. In present study curcumin showed alike anti-inflammatory effect in opposition to Nicotine in all curcumin treated groups through reducing levels of IL-6, IL-1β and TNF-α.
Intracellular calcium signaling controls scores basic cellular processes including proliferation, differentiation, and cellular motility [50]. Calcium levels are conserved at very low concentrations intracellularly via its removal to the extracellular environment and sequestration in the endoplasmic reticulum. As such, it is a powerful second messenger important in proliferation, differentiation, mitosis, and motility. In bone cells, an extracellular influx and intracellular release is rapidly activated by strain, pressure, and fluid flow [51][52][53]. In the current study Nicotine group has the lowest Calcium level. After nicotine treatment following curcumin administration calcium level slightly increased while dose dependent curcumin treatment groups has highest calcium level.
Thus, we suggest that curcumin is effective in reducing oxidative stress and bone lose induced by nicotine in rats.

Conclusion
As a result of our study, it was determined that there was a decrease in bone development and decrease in ossification rate in fetuses of pregnant rats exposed to nicotine. Different doses of curcumin were given to the rats against nicotine, and it was determined that the number of non-ossified bones decreased, and normal development was observed in bone development, especially in the high-dose curcumin-treated groups. As a conclusion curcumin therapy after nicotine administration markedly improved anatomical and biochemical findings and prohibited oxidative stress and inflammation. According to these data we propose that curcumin at the 100 and 150 mg may be used as a potential therapeutic agent to prevent bone lose induced by nicotine and our results will be beneficial in model studies which will be conducted on curcumin and nicotine.