Curcumin's protective role against fluoride-induced bone damage: Implications for pullet pathology and skeletal biomechanics

Qi-yong Zuo; Hai-qiang Meng; Mohammad Mehdi Ommati; Guo-dong Yang; Wen-peng Zhao; Jing Zhao; Bian-hua Zhou; Hong-wei Wang

doi:10.1016/j.psj.2025.104891

. 2025 Feb 6;104(3):104891. doi: 10.1016/j.psj.2025.104891

Curcumin's protective role against fluoride-induced bone damage: Implications for pullet pathology and skeletal biomechanics

Qi-yong Zuo ¹, Hai-qiang Meng ¹, Mohammad Mehdi Ommati ¹, Guo-dong Yang ¹, Wen-peng Zhao ¹, Jing Zhao ¹, Bian-hua Zhou ¹, Hong-wei Wang ^1,^⁎

PMCID: PMC11879666 PMID: 39970518

Abstract

This study aimed to investigate the effects of dietary fluoride (F) and curcumin (Cur) supplementation on the tibial biomechanical performance, histopathology, and behavior of pullets. Four dietary F levels (0, 400, 800, 1200 mg/kg) supplemented with CUR (0, 200 mg/kg) were used to create 8 experimental groups in Hyline Brown pullets. Behavioral study results showed that supplements of 1200 mg/kg F reduced the percentages of feeding, walking, standing, and preening behaviors while increasing the percentage of lying behavior (P < 0.05). This is associated with F-induced tibial elastic modulus, maximum stress, and stiffness coefficient were reduced and toughness coefficient was increased (P < 0.05). F-induced tibial cortical bone thickened, trabecular bone widened, and excessive accumulation of bone collagen fibers (P < 0.05) in the tibia explained the biomechanical properties reduction in Hyline Brown pullets. Additionally, the loss of antioxidant capacity was mediated by excessive F-accelerated pathological damage to the bone (P < 0.05). Supplementation with 200 mg/kg CUR alleviated abnormal behavior, expansion of the trabecular bone, accumulation of collagen fibers, and loss of antioxidant capacity (P < 0.05). In conclusion, F reduced the antioxidant level of the body, caused tibia histopathological damage, destroyed the tibia biomechanical properties, and caused abnormal behavior of pullets. Supplementation with 200 mg/kg CUR attenuates F-induced oxidative and tibia damage and rectifies abnormal behavioral traits.

Keywords: Antioxidants, Histopathology, Skeletal biomechanics, Behavior, Skeletal fluorosis

Graphical abstract

Introduction

Fluorine is a small, polar element commonly found in soil, water, and air as various compounds (Zuo et al., 2024). Epidemiological studies have shown that excessive environmental fluoride (F) exposure poses a serious public health problem in over 25 countries (Hou et al., 2024). More than 90 % of the F absorbed by the body accumulates in bones, causing lesions such as dental fluorosis, osteosclerosis, and osteoporosis, which threaten human health and livestock production. Studies report that bone F concentrations in animals from high F regions are more than tenfold higher than in regions with acceptable F levels (Yan et al., 2015; Johnston and Strobel, 2020; Rahim et al., 2022). Consequently, bones, as a primary F target, have garnered extensive scholarly attention. F accumulation in animal bones not only affects animal health but also enters the human food chain, posing a potential risk to human health.

F is highly reactive and possesses strong oxidizing properties. Excessive F intake can cause an imbalance between oxidative processes and the antioxidant defense system (Miao et al., 2017; 2019), elevating levels of free radicals and reactive oxygen species (ROS) while reducing the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and total antioxidant capacity (T-AOC). Additionally, the excessive synthesis of intracellular ROS induces cellular stress by upregulating the expression of protein kinases Mst1 and Mst2, thereby accelerating cell death (Ommati et al., 2024). Therefore, oxidative damage is considered a central mechanism in F-induced cell injury and death. Our research group demonstrated that excessive F promotes osteoclast proliferation, and increases the connectivity of trabeculae and the thickness of the growth plate, ultimately resulting in a high bone mass phenotype (Zhu et al., 2022; Jin et al., 2023). Curcumin (CUR), a phenolic compound with anti-inflammatory and antioxidant effects, was recently shown to inhibit osteoclast proliferation, reduce bone resorption, and improve bone microstructure by modulating the OPG/RANKL/RANK signaling pathway (Zia et al., 2021; Prasad et al., 2021; Li et al., 2023). At the enzyme level, CUR reduces the production of ROS by enzymes such as lipoxygenase/cyclooxygenase and xanthine dehydrogenase/oxidase. Additionally, it enhances the activity of SOD and peroxidase (POD), which serve as the first line of defense against oxygen-free radicals (Del Prado-Audelo et al., 2019). Could CUR supplementation resist F-induced oxidative damage and alleviate F-induced bone toxicity? This question warrants further investigation.

In poultry production, the onset of the laying period and the continuous increase in egg production led to increased mineral loss in the body. To compensate for this loss, producers often supplement poultry feed with phosphate minerals to maintain the physiological mineral requirements of the body. Phosphate minerals contain 2 %-3 % F, and improper defluoridation processes or the use of substandard feed can result in F levels in poultry feed exceeding 350 mg/kg, posing a potential threat to poultry health and production (Ranjan and Ranjan, 2015). Does the accumulation of excessive F in bones cause pathological damage to bone tissues and behavioral changes in poultry? This study examines F accumulation in bones and its effects on bone biomechanics and histopathology through CUR supplementation in pullet feed. The study also investigates the role of CUR in alleviating skeletal fluorosis, providing a theoretical and practical basis for preventing and treating fluorosis.

Materials and methods

The experimental design was approved by the Institutional Animal Experiment Committee of Henan University of Science and Technology, China (HAUST-024-P0818002).

Experimental design

A total of 288 Hyline Brown pullets were randomly assigned to 8 groups, with 3 replicates per group containing 12 pullets. The basic diet was prepared according to the National Research Council's guidelines. The experimental design was structured as follows: pullets in the control group were given a standard diet, whereas those in the F groups were provided with diets containing sodium fluoride (NaF) at 400, 800, or 1200 mg/kg, measured as F ion. In addition to NaF, CUR (200 mg/kg) was added to establish an F-exposure model with CUR supplementation. The experimental groups included: the control group, F 400 group, F 800 group, F 1200 group, CUR control group, CUR F 400 group, CUR F 800 group, and CUR F 1200 group. Behavioral observations were analyzed in the 5th, 11th, and 17th weeks. Blood was collected for serum separation to assess the antioxidant index before the pullets were euthanized in the 17th week. Tibial tissues were collected for analysis of X-ray, bone parameters, biomechanics, and morphology.

Behavioral observation

After marking the pullets with biological dyes, behavioral characteristics were recorded using an Ant Smart Camera at the 5th, 11th, and 17th weeks, from 8:00–10:00 am. to 2:00–4:00 pm. The data were analyzed by the Observer XT 12 behavior analysis software (Noldus Information Technology BV, the Netherlands). Behavioral variables (Feeding, Drinking, Walking, Standing, Lying, Preening, Stretching) were evaluated according to the study by Ma et al. (2020).

Antioxidant capacity and Ca and P content analysis

To analyze the antioxidant capacity in pullets, serum samples were isolated by centrifugation at 3000 rpm for 10 min at 4°C following blood coagulation. Following this, malondialdehyde (MDA; catalog number: A003-1-1), SOD (A001-2-2), calcium (Ca; C004-2-1), and T-AOC (A015-3-1) were analyzed using an enzyme-labeled instrument. Catalase (CAT; A007-1-1), phosphorus (P; C006-1-1), and GSH-Px (A005-1-2) were analyzed with a spectrophotometer. The commercial kits were sourced from Nanjing Jiancheng Bioengineering Institute, Nanjing, China.

Tibia X-ray and parameters

The relative bone mass of tibial tissues in pullets was measured using digital radiography. At the same time, skeletal lesions in the pullets’ claw were observed using DR (digital radiography). The tibial weight of the left leg was measured using an analytical balance. The tibial length, transverse diameter, and longitudinal diameter at the midpoint were measured using a vernier caliper.

Histological analysis

When tibiae were demineralized using EDTA decalcification fluid, they were dehydrated in a gradient series of alcohol and cleared with xylene. Afterward, the specimens were embedded in paraffin and sectioned into 4 µm slices. Hematoxylin and Eosin (H&E) staining and Masson staining were performed according to the manufacturer's protocols. We acquired scanned images on H&E sections using a digital scanner. The thicknesses of cortical bones were measured with CaseViewer software (The Digital Pathology Company). The areas of trabecular bone and bone collagen fibers were quantified by Image J analysis software.

Determination of bone F content

After opening the pullets’ tibia, the bone marrow was removed from the cavity and rinsed with PBS. The specimens were dried at 105°C for 4 h and then weighed. Before ashing, the dried specimens were heated in a fume hood in a smoke-free environment. Next, the specimens were ashed for 6 h at 550°C. Finally, the ashed specimens were immersed in hydrochloric acid to remove carbonate anions. F content in the solution was measured using a potentiometric method with an ion-selective electrode (Leici, PF-1 01, Shanghai, China) (Del Razo et al., 1993).

Tibia biomechanics determination

The tibia underwent a three-point bending test using the Shimadzu AGS-X universal electronic testing machine to evaluate its biomechanical properties. The loading speed was set at 5 mm/min, with a support span of 80 mm. Biomechanical parameters were analyzed using TRAPEZIUM LITE X software.

Statistical analysis

Data analysis was performed using two-way and three-way analysis of variance (ANOVA) in SPSS 22.0 (SPSS Inc., Chicago, IL). Data are presented as means, mean standard errors (SEM), and P-values. Differences between treatments were assessed using Tukey's multiple comparison test, with significance set at P < 0.05. Pearson's correlation analysis was conducted using Origin 2022 software.

Results

Effects of CUR on the behavior of F-exposed pullets

The effects of F levels and CUR supplementation at the 5th, 11th, and 17th weeks on the behavior of pullets are shown in Table 1. Week, F and CUR had significant interaction on feeding and drinking behaviors (P = 0.001). Pullets receiving 200 mg/kg CUR in the 5th week exhibited a higher percentage of feeding behavior compared to other groups (P < 0.05). Regardless of the timing or CUR supplementation dietary F levels of 800 and 1200 mg/kg inhibited the feeding behavior of pullets (P < 0.05). CUR supplementation at 200 mg/kg increased the percentage of feeding behavior (P < 0.05). Significant interactions among week, F, and CUR were also observed for preening behavior (P < 0.05). In the 11th week, pullets receiving 200 mg/kg CUR showed a higher percentage of preening behavior compared to other treatment groups (P < 0.05). From the point of main effect, the 11th week showed a higher percentage of preening behavior than the 5th and 17th weeks (P < 0.05). Similarly, increased F levels (400, 800, and 1200 mg/kg) significantly elevated the percentage of preening behavior compared to the control group (P < 0.05), with the 1200 mg/kg group showing the lowest percentage. No significant interactions among week, F, and CUR were found for walking (P = 0.816), standing (P = 0.945), lying (P = 0.381), and stretching (P = 0.999) behaviors. The main effects indicated that dietary F levels of 400, 800, and 1200 mg/kg increased lying behavior percentages compared to control groups (P < 0.05). Meanwhile, these F levels decreased the percentages of walking, standing, and stretching behaviors compared to the 0 mg/kg F diet (P < 0.05). Supplementation with 200 mg/kg CUR reduced the percentage of lying behavior (P < 0.05).

Table 1.

Effects of CUR on the behavior of F-exposed pullets.

Week	F (mg/kg)	CUR (mg/kg)	Feeding (%)	Drinking (%)	Walking (%)	Standing (%)	Lying (%)	Preening (%)	Stretching (%)
5th	0	0	23.07^b	7.42^abc	15.39^a	12.61^fghi	29.15^ijk	10.95^gh	1.35^ab
5th	400	0	24.50^ab	7.67^abc	12.92^abc	13.71^efg	29.41^ij	10.67^ghi	1.03^abcd
5th	800	0	17.60^cd	4.03^efg	10.86^abcde	7.08^kl	51.75^ced	7.67^lm	0.75^abcd
5th	1200	0	14.20^ef	4.32^efg	10.29^bcdefg	4.55^l	59.25^a	6.58^m	0.82^bcd
5th	0	200	26.89^a	6.22^cd	15.35^a	13.86^efg	23.53^m	12.73^fg	1.43^a
5th	400	200	26.52^a	6.33^c	13.79^ab	14.61^ef	25.20^klm	12.47^fg	1.08^abcd
5th	800	200	18.82^c	4.83 ^de	11.95^abcd	10.01^hijk	45.50^fg	8.38^jklm	0.89^abcd
5th	1200	200	15.09^de	4.65^ef	10.73^abcde	8.72^jk	51.67^cde	8.27^klm	0.87^abcd
11th	0	0	17.08^cd	8.78^a	12.55^abcd	12.87^fghi	27.69^jkl	19.70^c	1.34^abc
11th	400	0	17.25^cd	6.57^c	10.87^abcde	14.45^ef	30.61^hij	19.05^c	1.21^abcd
11th	800	0	10.58^ghi	6.72^c	8.89^cdefgh	13.16^fgh	48.21^ef	11.47^gh	0.99^abcd
11th	1200	0	9.92^hij	4.82^de	9.75^cdefgh	9.45^ijk	57.05^ab	8.11^lm	0.87^abcd
11th	0	200	15.52^de	8.34^ab	13.75^ab	12.26^fghij	23.64^lm	29.49^a	1.38^ab
11th	400	200	17.47^cd	7.03^bc	11.32^abcd	13.16^fgh	24.21^lm	25.63^b	1.21^abcd
11th	800	200	12.82^efg	6.63^c	9.24^bcdefgh	14.52^ef	42.21^g	14.52^ef	1.09^abcd
11th	1200	200	11.43^fgh	6.61^c	10.95^abcde	10.67^ghij	51.29^de	9.54^ijkl	0.93^abcd
17th	0	0	9.74^hij	2.95^g	10.59^bcdef	30.50^a	28.84^ijk	17.38^cd	1.24^abcd
17th	400	0	8.01^ijk	3.16^g	7.87^defgh	27.23^ab	34.69^h	17.49^cd	0.89^abcd
17th	800	0	5.43^k	3.56^efg	6.21^efgh	20.65^c	55.96^ab	9.44^ijkl	0.76^cd
17th	1200	0	5.45^k	3.67^efg	5.96^fgh	16.97^de	58.29^a	7.97^lm	0.69^d
17th	0	200	10.11^ghi	2.97^g	10.69^abcdef	28.30^ab	30.10^ij	16.61^de	1.22^abcd
17th	400	200	10.87^gh	3.27^fg	9.84^bcdefgh	25.68^b	32.65^hi	16.57^de	0.94^abcd
17th	800	200	7.26^jk	3.97^efg	5.17^h	19.75^cd	53.85^bcd	11.29^gh	0.73^d
17th	1200	200	6.69^k	3.45^efg	5.79^gh	17.14^cde	55.71^abc	10.55^hijk	0.68^d
SEM			1.31	0.37	0.58	1.38	2.68	1.19	0.05
Main effect
Week
5th			20.84^a	5.68^b	12.66^a	10.64^c	39.43^b	9.71^c	1.03^a
11th			10.98^b	6.94^a	10.91^b	12.57^b	38.11^c	17.19^a	1.12^a
17th			7.95^c	3.37^c	7.76^c	23.28^a	43.76^a	13.41^b	0.89^b
SEM			3.89	1.05	1.43	3.93	1.71	2.16	0.07
F (mg/kg)
0			17.07^a	6.11^a	13.05^a	18.40^a	27.16^d	17.81^a	1.32^a
400			17.44^a	5.67^b	11.10^b	18.14^a	29.46^c	16.98^b	1.06^b
800			12.09^b	4.95^c	8.72^c	14.20^b	49.58^b	10.46^c	0.87^c
1200			10.46^c	4.58^d	8.91^c	11.25^c	55.54^a	8.50^d	0.81^c
SEM			1.76	0.35	1.02	1.71	7.12	2.33	0.11
CUR (mg/kg)
0			13.57^b	5.30	10.18	15.27	42.57^a	12.21^b	0.99
200			14.96^a	5.36	10.71	15.72	38.29^b	14.67^a	1.03
SEM			0.70	0.03	0.27	0.23	2.14	1.23	0.02
P-values
Week			< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001
F			< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001
CUR			< 0.001	0.647	0.149	0.102	< 0.001	< 0.001	0.368
Week × F × CUR			0.001	0.001	0.816	0.945	0.381	< 0.001	0.999

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CUR, Curcumin; F, fluoride; SEM, standard error of the mean.

^a–mMeans with different letters in the column are significantly different (P < 0.05).

Effects of CUR on the bone morphology of F-exposed pullets

Excessive F caused metatarsal sclerosis in the pullet compared to the control group, particularly in the F 1200 group. Supplementation with 200 mg/kg CUR alleviated this phenomenon (Fig. 1A). Excess F increased the relative bone mass of the tibia by 37.55 % in the F 400, 32.28 % in the F 800, and 30.04 % in the F 1200 groups (P < 0.05, Fig. 1B and C).

Fig 1 — Effects of F exposure on claw and bone structure. (A) X-ray diffraction analysis of the claw of pullets. Optical (left) and X-ray (right) image of pullets’ claw. Red arrows represent abnormal claws. (B) X-ray diffraction analysis of the claw of pullets. (C) The average area of bone trabecular analysis in HE staining. Data were shown as mean ± SEM (n = 6). Black arrows represent normal osteocytes. Red arrows represent porous structures in the bone cortex. Blue arrows represent cracks in the trabecular bone. ^a-c Means with different letters in the column are significantly different (P < 0.05).

Effects of CUR on the tibia histopathology of F-exposed pullets

As shown in Fig. 2, the cortical bone structure of pullets' tibiae was intact, with an orderly arrangement and complete morphology of bone cells in the control group (Black arrows, Fig. 2A). Compared to the control group, the cortical bone thickness and F ion content in the tibia were increased in all F-treatment groups (P < 0.05, Fig. 2A and D), and a large amount of porous structure was observed in the F800 and F1200 groups. Supplementation with 200mg/kg CUR increased the thickness of cortical bone by 15.13 % in the CUR F 1200 group (P < 0.05, Fig. 2B). Supplementation with CUR effectively reversed the excessive F-induced increase of the porous structure in the CUR F 800 and the CUR F 1200 groups (Red arrows, Fig. 2A).

The trabecular bone of pullets' tibiae had an intact structure and clear orientation in the control group. Compared to the control group, the average area of the trabeculae was increased in all F treatment groups (P < 0.05, Fig. 2A and C). We also found that excess F increased trabecular cracks in the F 800 and F 1200 groups (Blue arrows, Fig. 2A). Adding 200mg/kg CUR reduced the average area of trabeculae and alleviated the increase of bone trabecular cracks in the CUR F 800 and CUR F 1200 groups (P < 0.05, Fig. 2A and C).

Effects of CUR on the bone collagen fibers of F-exposed pullets

As shown in Fig. 3, the percentage of collagen fibers area in the pullet tibia was increased in the F 800 and F 1200 groups (P < 0.05, Fig. 3A and B). Compared with the F treatment groups, adding CUR could alleviate the trend of excessively increasing the percentage of collagen fiber area (P < 0.05, Fig. 3A and B).

Effects of CUR on the tibia parameters and biomechanical of F-exposed pullets

As shown in Table 2, there was a significant interaction between the dietary F levels and CUR supplementation on the transverse diameter of the tibia (P = 0.005) and tibial weight (P = 0.013). In the control group, the transverse diameter of the tibia and tibial weight were lower than in other treatment groups (P < 0.05). Similarly, the F1200 group inhibited tibial length compared to control, F400, and F800 groups (P < 0.05). Supplementation with 200 mg/kg CUR increased the transverse diameter of the tibia and tibial weight in pullets (P < 0.05).

Table 2.

The effect of CUR on F-exposed pullet bone parameters.

Treatments	F (mg/kg)	CUR (mg/kg)	Tibia length (mm)	Transverse diameter of tibia midpoint (mm)	Longitudinal diameter of tibial midpoint (mm)	Tibial weight (g)	Cross-sectional moment of inertia (mm⁴)
CON	0	0	125.92^a	6.46^b	7.73	10.69^b	69.38^c
F400	400	0	124.00^a	6.92^ab	8.32	11.80^ab	90.04^ab
F800	800	0	122.99^a	6.83^ab	7.87	12.17^a	95.03^a
F1200	1200	0	116.65^b	6.75^ab	7.77	11.16^bc	84.06^ab
CUR	0	200	125.59^a	7.29^a	8.26	12.47^a	81.70^b
CUR F400	400	200	124.17^a	6.83^ab	8.25	12.61^a	89.54^ab
CUR F800	800	200	124.16^a	7.15^a	7.71	12.49^a	96.53^a
CUR F1200	1200	200	119.07^b	6.68^ab	7.70	12.03^ab	92.23^ab
SEM			1.15	0.09	0.10	0.24	3.12
Main effect
F (mg/kg)
	0		125.75^a	6.87	7.99^ab	11.58^b	75.54
	400		124.08^b	6.88	8.28^a	12.20^a	89.79
	800		123.58^b	6.99	7.79^b	12.33^a	95.78
	1200		117.86^c	6.72	7.73^b	11.59^b	88.14
SEM			1.72	0.06	0.12	0.20	4.25
CUR (mg/kg)
		0	122.39	6.74^b	7.92	11.46^b	84.62
		200	123.24	6.99^a	7.98	12.40^a	90.00
SEM			0.42	0.08	0.03	0.47	2.69
P-values
F			< 0.001	0.273	0.029	< 0.001	< 0.001
CUR			0.092	0.014	0.669	< 0.001	0.072
F × CUR			0.23	0.005	0.280	0.013	0.386

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CUR, Curcumin; F, fluoride; SEM, standard error of the mean;.

^a-cMeans with different letters in the column are significantly different (P < 0.05).

The biomechanical results of the pullets' tibias are shown in Table 3. There was no significant interaction between dietary F levels and CUR supplementation on the pullets ' elastic modulus (P = 0.616), maximum stress (P = 0.542), maximum strain (P = 0.689), rigidity coefficient (P = 0.350), and toughness coefficient (P = 0.070). The main effects indicated that dietary F levels of 400, 800, and 1200 mg/kg decreased the elastic modulus, maximum stress, and rigidity coefficient (P < 0.05), with the lowest values observed in the F1200 treatment group: 32.09 MPa, 43.43 MPa, and 363.76 N·mm², respectively. Supplementation with 200 mg/kg CUR increased the pullets' elastic modulus and maximum strain (P < 0.05). Based on Table 2, Table 3, the damaging effects of F on bone development parameters and biomechanical properties are dose-dependent, and supplementation with 200 mg/kg CUR can improve the damage caused by F to the bones.

Table 3.

The effect of CUR on F-exposed pullets tibiabiomechanical performance.

Treatments	F (mg/kg)	CUR (mg/kg)	Elastic modulus (MPa)	Maximum stress (MPa)	Maximum strain (%)	Rigidity coefficient (N·mm²⁾	Toughness coefficient (mm·N^-1)
CON	0	0	46.38^ab	62.96^a	2.06^c	489.67^ab	0.04^d
F400	400	0	39.05^bcd	56.67^abc	4.18^ab	486.60^ab	0.08^bc
F800	800	0	37.19^cd	47.67^bc	3.80^ab	428.70^ab	0.10^ab
F1200	1200	0	32.09^d	43.43^c	3.30^b	363.76^b	0.12^a
CUR	0	200	53.12^a	61.51^ab	2.26^c	560.65^a	0.06^c
CUR F400	400	200	44.27^bc	49.65^abc	4.31^a	450.34^ab	0.10^ab
CUR F800	800	200	39.64^bcd	49.85^abc	4.30^a	449.03^ab	0.10^a
CUR F1200	1200	200	35.36^d	43.65^c	3.80^ab	397.69^b	0.11^a
SEM			2.38	2.69	0.32	21.42	0.01
Main effect
F (mg/kg)
	0		49.75^a	62.23^a	2.16^c	525.16^a	0.05^d
	400		41.66^b	53.16^b	4.24^a	468.47^ab	0.09^c
	800		38.41^c	48.76^bc	4.05^ab	438.86^bc	0.10^b
	1200		33.72^c	43.54^c	3.55^b	380.72^c	0.11^a
SEM			3.70	3.96	0.47	25.32	0.01
CUR (mg/kg)
		0	38.67^b	52.68	3.34^b	442.18	0.08
		200	42.67^a	51.16	3.67^a	464.42	0.09
SEM			2.00	0.76	0.17	11.12	0.01
P-values
F			< 0.001	< 0.001	< 0.001	< 0.001	< 0.001
CUR			0.001	0.517	0.021	0.295	0.070
F × CUR			0.616	0.542	0.689	0.350	0.070

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CUR, Curcumin; F, fluoride; SEM, standard error of the mean;.

^a-dMeans with different letters in the column are significantly different (P < 0.05).

Effects of CUR on the serum indicators of F-exposed pullets

Table 4 shows the results of the antioxidant indicators and Ca and P content in the pullet' serum. There was a significant interaction between dietary F levels and CUR on the concentration of SOD in the pullets’ serum (P = 0.026). In the F 1200 group, the SOD content was the lowest at 226.35 U/mL (P < 0.05). Supplementation with 200 mg/kg CUR increased the pullets’ concentration of SOD (P < 0.05). There was no significant interaction between dietary F and CUR levels on GSH-Px, CAT, T-AOC, MDA, Ca, and P concentrations (P > 0.05). The main effects indicated that dietary F levels of 400, 800, and 1200 mg/kg decreased GSH-Px, CAT, T-AOC, and Ca concentrations in the pullets' serum and increased the concentrations of MDA and P (P < 0.05). Supplementation with 200mg/kg CUR increased the levels of CAT, and T-AOC and decreased the concentrations of MDA and P (P < 0.05).

Table 4.

The effect of CUR on F-exposed pullet serum indicators.

Treatments	F (mg/kg)	CUR (mg/kg)	GSH-Px(U/mL)	CAT (U/mL)	T-AOC (U/mL)	SOD (U/mL)	MDA (mmol/ml)	Ca (mmol/L)	P (mmol/L)
CON	0	0	717.81^b	4.25^d	4.34^bc	326.87^bc	4.07^cd	5.08^a	1.83^c
F400	400	0	640.95^d	3.85^cd	3.26^e	307.32^c	4.85^bc	4.41^cd	1.98^cd
F800	800	0	524.09^f	3.25^bc	3.53^de	267.71^d	5.75^ab	4.21^d	2.32^bc
F1200	1200	0	423.57^h	2.85^a	2.77^f	226.35^e	5.98^a	4.08^d	2.97^a
CUR	0	200	750.86^a	4.51^d	5.34^a	406.74^a	3.21^c	4.96^a	1.75^c
CUR F400	400	200	674.21^c	4.13^d	4.49^b	388.12^a	3.95^cd	4.79^ab	1.82^cd
CUR F800	800	200	568.92^e	3.86^cd	4.45^b	343.43^b	4.59^c	4.55^abc	1.94^c
CUR F1200	1200	200	481.61^g	3.42^b	3.89^cd	271.74^d	4.67^c	4.35^cd	2.46^b
SEM			16.11	0.20	0.29	21.83	0.32	0.13	0.15
Main effect
F (mg/kg)
	0		734.33^d	3.14^d	4.84^a	366.80^d	3.64^c	5.01^a	1.79^c
	400		657.58^c	3.55^c	3.87^b	347.72^c	4.40^b	4.60^b	1.90^c
	800		546.50^b	3.99^b	3.99^b	305.57^b	5.17^a	4.38^bc	2.13^b
	1200		452.59^a	4.38^a	3.33^c	249.04^a	5.32^a	4.21^c	2.71^a
SEM			61.87	0.21	0.31	26.10	0.39	0.17	0.21
CUR (mg/kg)
		0	576.60	3.55^b	3.47^b	282.06^b	5.16^a	4.44	2.27^a
		200	618.89	3.98^a	4.54^a	352.50^a	4.11^b	4.66	1.99^b
SEM			21.15	0.14	0.54	35.22	0.53	0.11	0.14
P-values
F			< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001
CUR			< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	0.014	< 0.001
F × CUR			0.254	0.147	0.485	0.026	0.739	0.157	0.162

Open in a new tab

CUR, Curcumin; F, fluoride; SEM, standard error of the mean;.

^a-hMeans with different letters in the column are significantly different (P < 0.05).

Correlation analysis between biomechanics and behavior

As shown in Fig. 4, Pearson correlation analysis was conducted to explore the relationship between the locomotor (walking, standing, and lying) behavior and biomechanical performance of 17th week pullets. The walking behavior showed a positive correlation with the elastic modulus (r = 0.75, P < 0.05) and maximum stress (r = 0.64, P < 0.05), but a negative correlation with maximum strain (r = - 0.58, P < 0.05) and toughness coefficient (r = - 0.81, P < 0.05). Similarly, standing behavior was positively correlated with the elastic modulus (r = 0.68, P < 0.05), maximum stress (r = 0.71, P < 0.05), and rigidity coefficient (r = 0.63, P < 0.05), but negatively correlated with toughness coefficient (r = - 0.98, P < 0.05). The lying behavior exhibited a negative correlation with the elastic modulus (r = - 0.69, P < 0.05), maximum stress (r = - 0.68, P < 0.05), and rigidity coefficient (r = - 0.55, P < 0.05), whereas it showed a positive correlation with toughness coefficient (r = 0.90, P < 0.05). After the supplementation of CUR, the standing behavior showed a negative correlation with maximum strain (r = - 0.48, P < 0.05), while the lying behavior exhibited a positive correlation with maximum strain (r = 0.41, P < 0.05).

Discussion

Animals primarily ingest F through the digestive tract. In the stomach, F reacts with gastric acid to form hydrofluoric acid, which severely corrodes and damages gastric epithelial cells, destroying the gastric mucosa. Numerous studies demonstrated that excessive F can damage the mechanical barrier of the intestine and disrupt the intestinal microenvironment, inhibiting digestion, and absorption capabilities, thereby reducing feed intake (Jin et al., 2022; Gao et al., 2023; Li et al., 2024). This study found that excessive F resulted in a significant reduction in the percentage of feeding behavior. We hypothesize that this phenomenon is closely related to the damage inflicted on the gastric mucosa of pullets due to excessive F. Preening is a positive behavior. This study found that F decreased grooming behavior during the same week. Studies have confirmed that excessive F could cause anxiety behavior in mice, which is manifested as slow movement (Li et al., 2019). F may inhibit hormones or neurotransmitters, leading to the inhibition of preening behavior. Our research found that feeding, drinking, and preening behavior are influenced by the combination of time, F, and CUR. CUR has anti-inflammatory and antioxidation properties and may extent alleviate F-induced digestive tract and nervous system damage, thereby mitigating the reduction in feeding, drinking, and preening behavior induced by F. Therefore, it can be inferred that CUR plays a crucial role in ameliorating F-induced behavioral abnormalities.

Further behavioral research showed that excessive F reduced the percentage of walking and standing behaviors while increasing lying behavior in pullets. This suggests that F exposure inhibited the motor behaviors of the pullets. Standing and lying behaviors in animals are closely related to leg health (Güz et al., 2020), and prolonged lying may indicate an adaptive response to leg conditions. F, a potent skeletal toxin, can convert bone mineral hydroxyapatite into fluorapatite, altering the crystal structure and biological properties of bone (Johnston and Strobel, 2020), leading to severe motor dysfunction (Wang et al., 2012). Accordingly, the biomechanical properties of pullets were measured following F exposure. Results indicated that the elastic modulus, maximum stress, and stiffness coefficient were reduced following F exposure, while the toughness coefficient increased. After supplementing with CUR, the F-induced decrease in elastic modulus can be alleviated. The increase in elastic modulus can enhance the load-bearing capacity of the bones and reduce the risk of fractures. Correlation analysis revealed strong associations between biomechanical properties and behaviors. Changes in biomechanical properties due to excessive F are the underlying cause of motor dysfunction in pullets.

Bone is a highly active tissue, and the essential physiological remodeling processes in healthy animals create a window for fluorosis development. Excessive F disrupts the calcium-phosphorus balance, leading to abnormal bone remodeling and metabolic activities in pullets. With 1200 mg of F added to the feed, tibial F content reached 8900 mg/kg, 34.2 times that of the control group. Similarly, the present study observed a decrease in blood calcium and an increase in blood phosphorus after F exposure. In the bones of pullets, F combines with hydroxyapatite and calcium to form fluorapatite and calcium fluoride, resulting in F accumulation in bone, which may account for the decrease in serum calcium.

Bones in the body comprise cortical and trabecular bone, which exhibit different responses to stress. Mechanically, cortical bone provides structural strength and stiffness (Hemmatian et al., 2017), while trabecular bone absorbs energy and transfers pressure under external stress (Alcorta-Sevillano et al., 2020). An imbalance in cortical and trabecular bone distribution can alter biomechanical properties. Our findings showed that excessive F unexpectedly increased cortical bone thickness in the tibia of pullets, without a corresponding increase in maximum stress or stiffness. Light microscopy revealed numerous porous structures in the cortical bone, likely resulting from an imbalance in cortical bone remodeling induced by excessive F. Despite increased cortical thickness, porous structures altered biomechanical properties, compromising integrity. Additionally, excessive F increased trabeculae, with some regions fusing into patches and developing cracks. The presence of cracks prevents trabeculae from effectively responding to tibial loading, hindering load transfer to the cortical bone and reducing biomechanical performance.

Bone mineralization and collagen fiber organization are fundamental to healthy bone biological function. Collagen fibers serve as the structural framework, playing a crucial role in bone formation and providing a template for mineralization (Schaffler and Burr, 1988). This study confirms that excessive F leads to collagen fiber accumulation within trabeculae, suggesting that increased collagen fiber quantity may not effectively enhance biomechanical performance. Research indicates that abnormal orientation and disordered arrangement of collagen fibers can compromise bone biomechanical properties (Barceló et al., 2023). Previous research demonstrated that excessive F caused Type I collagen fiber disarray in rat femurs, aligning with the findings of this study (Zhu et al., 2022). Thus, F-induced disruptions in bone mineralization and collagen fiber accumulation are primary factors contributing to decreased bone biomechanical performance.

Oxidative stress is widely recognized as the primary mechanism underlying F-induced systemic toxicity (Jaiswal et al., 2020). As a reactive small molecule, F can disrupt oxygen metabolism, reduce cellular antioxidant enzyme activity, impair antioxidant defenses, and lead to free radical accumulation and severe cellular damage. Previous research confirmed that ROS-mediated intracellular oxidative stress impairs mitochondrial respiratory function, promoting cytochrome c release, which then triggers a caspase cascade, resulting in apoptosis (Wang et al., 2019). This study observed that excessive F reduced SOD, CAT, and GSH-px activities and GSH content in pullet serum while increasing MDA content. This reduction in antioxidant capacity is a critical mechanism through which F affects pullet bone biomechanics and behavior. CUR, an antioxidant, can neutralize superoxide anions, peroxynitrite, and nitric oxide (Immediata et al., 2022). CUR supplementation in pullet feed reversed F-induced oxidative damage, improved tibial development, and resisted tibial pathology and morphological changes, ultimately enhancing bone biomechanics and pullet behavior. Given the lack of targeted drugs for fluorosis, CUR has been used as a growth-promoting additive in aquaculture (Ashry et al., 2021) and pig production (Niu et al., 2019). Findings indicate that CUR counteracts the skeletal damage caused by excessive F in pullets.

In conclusion, F reduced the body's antioxidant level, caused tibia histopathological damage, destroyed the tibia biomechanical properties, and caused abnormal behavior of pullets. Supplementation with 200mg/kg CUR attenuates F-induced oxidative and tibia damage and rectifies abnormal behavioral traits.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant Nos.·32102740 and 32373084), Natural Science Foundation of Henan Province (Grant No. 242300420146) and Luoyang Rural Revitalization Public Welfare Special Project (Grant No. 2302030A).

Contributor Information

Qi-yong Zuo, Email: zuoqy@stu.haust.edu.cn.

Hai-qiang Meng, Email: 230320181021@stu.haust.edu.cn.

Mohammad Mehdi Ommati, Email: mehdi_ommati@outlook.com.

Guo-dong Yang, Email: ygd2002@126.com.

Wen-peng Zhao, Email: zhaowp@haust.edu.cn.

Jing Zhao, Email: hkdzhao1230@haust.edu.cn.

Bian-hua Zhou, Email: zhoubh@haust.edu.cn.

Hong-wei Wang, Email: wanghw@haust.edu.cn.

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[bib0004] Del Prado-Audelo M.L., Caballero-Florán I.H., Meza-Toledo J.A., Mendoza-Muñoz N., González-Torres M., Florán B., Cortés H., Leyva-Gómez G. Formulations of curcumin nanoparticles for brain diseases. Biomolecules. 2019;9:56. doi: 10.3390/biom9020056. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib0007] Güz B.C., Molenaar R., de Jong I.C., Kemp B., van Krimpen M., van den Brand H. Effects of eggshell temperature pattern during incubation on tibia characteristics of broiler chickens at slaughter age. Poult. Sci. 2020;99:3020–3029. doi: 10.1016/j.psj.2019.12.042. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib0009] Hou L., Dong H., Zhang E., Lu H., Zhang Y., Zhao H., Xing M. A new insight into fluoride induces cardiotoxicity in chickens: involving the regulation of PERK/IRE1/ATF6 pathway and heat shock proteins. Toxicology. 2024;501 doi: 10.1016/j.tox.2023.153688. [DOI] [PubMed] [Google Scholar]

[bib0010] Immediata V., Ronchetti C., Spadaro D., Cirillo F., Levi-Setti P.E. Oxidative stress and human ovarian response-from somatic ovarian cells to oocytes damage: a clinical comprehensive narrative review. Antioxidants. 2022;11:1335. doi: 10.3390/antiox11071335. (Basel) [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib0012] Jin Y., Gao X.Y., Zhao J., Tian W.S., Zhang Y.L., Tian E.J., Zhou B.H., Wang H.W. Estrogen deficiency aggravates fluoride-induced small intestinal mucosa damage and junctional complexes proteins expression disorder in rats. Ecotoxicol. Environ. Saf. 2022;246 doi: 10.1016/j.ecoenv.2022.114181. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Curcumin's protective role against fluoride-induced bone damage: Implications for pullet pathology and skeletal biomechanics

Qi-yong Zuo

Hai-qiang Meng

Mohammad Mehdi Ommati

Guo-dong Yang

Wen-peng Zhao

Jing Zhao

Bian-hua Zhou

Hong-wei Wang

Abstract

Graphical abstract

Introduction

Materials and methods

Experimental design

Behavioral observation

Antioxidant capacity and Ca and P content analysis

Tibia X-ray and parameters

Histological analysis

Determination of bone F content

Tibia biomechanics determination

Statistical analysis

Results

Effects of CUR on the behavior of F-exposed pullets

Table 1.

Effects of CUR on the bone morphology of F-exposed pullets

Fig. 1.

Effects of CUR on the tibia histopathology of F-exposed pullets

Fig. 2.

Effects of CUR on the bone collagen fibers of F-exposed pullets

Fig. 3.

Effects of CUR on the tibia parameters and biomechanical of F-exposed pullets

Table 2.

Table 3.

Effects of CUR on the serum indicators of F-exposed pullets

Table 4.

Correlation analysis between biomechanics and behavior

Fig. 4.

Discussion

Declaration of competing interest

Acknowledgments

Contributor Information

References

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