Studies on the Effect of Curcumin and Quercetin in the Liver of Male Albino Rats Exposed to Gamma Irradiation

doi:10.21203/rs.3.rs-2917796/v1

Ionizing radiation produces deleterious effects on living organisms. The present investigation has been carried out to study the prophylactic as well as the therapeutic effects of treated rats with quercetin (Quer) and curcumin (Cur) which are two medicinal herbs known for their antioxidant activities against damages induced by whole-body fractionated gamma irradiation. Exposure of rats to whole-body gamma irradiation induced a significant decrease in erythrocyte (RBC), leukocyte (WBCs), platelets count (Plt), hemoglobin concentration (Hb), hematocrit (Hct %), mean erythrocyte hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and mean erythrocyte volume (MCV), high increase in plasma Thiobarbituric acid reactive substances (TBARS), non-significant statistical decrease in the mean value of serum glutathione (GSH), with a significant increase in plasma alanine transferase (ALT), aspartate transferase (AST), alkaline phosphates (ALP), serum total protein, serum total cholesterol levels, total triglycerides levels, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) levels with marked histological changes and structural changes measured by Fourier Transform Infrared (FTIR). Applying both quercetin and curcumin pre- and post-exposure to gamma-radiation revealed a remarkable improvement in all the studied parameters. The cellular damage by gamma radiation is greatly mitigated by the coadministration of curcumin and quercetin before radiation exposure.

Biological sciences/Biochemistry

Biological sciences/Biophysics

Biological sciences/Cancer

Biological sciences/Drug discovery

Health sciences/Medical research

Health sciences/Molecular medicine

Exposure to ionizing radiation (IR) is inevitable since over 80% of the total average exposure comes from natural sources. Therefore, there is a pressing need to protect humans against the effects of ionizing radiation. The liver, as a very important detoxification organ in the body, is vulnerable to the deleterious effects of IR. Attempts to protect against the harmful effects of ionizing radiation by pharmacological intervention were made as early as 1949. Living organisms are always exposed to oxidative stress and toxic hazards from radiation, pollution, toxins, and others. The natural and artificial sources of ionizing radiation produce direct and indirect damage to cells. Increased reactive oxygen species (ROS) generated from the stress condition of IR cause lipid, DNA, and protein damage [1, 2].

Ionizing radiation is known to generate ROS in irradiated tissue. Because most tissues contain 80% water, the major radiation damage is due to the aqueous free radicals, generated by the action of radiation on water. Hydroxyl radicals (•OH), are considered the most damaging of all free radicals generated in organisms [3]. The hydroxyl radical, produced during oxidative stress or radiation injury, induces a breakage in the DNA single strand [4], and DNA damage induces cell death [5]. Consequently, several processes for DNA repair are activated to counteract the DNA strand breaks caused by oxidative stress. Ionizing radiation induces different types of DNA damage in bone marrow cells that may remain unrepaired. In this situation, unrepaired DNA damage may lead to cell death or genomic instability [6]. Therefore, the risk of death or hematopoietic malignancies threatens the exposed people.

Curcumin is a major yellow pigment in turmeric ground rhizome of Curcuma Longa Linn which is used widely as a spice and coloring agent in several foods such as curry, mustard, and potato chips as well as cosmetics and drugs [7, 8]. The antioxidant activity of curcumin arises mainly from the scavenging of several biologically relevant free radicals that are produced during physiological processes [9, 10]. Quercetin, a unique bioflavonoid, is found in fruits, vegetables, grains, bark roots, stems, flowers, tea, and others [11]. It is considered a powerful antioxidant flavonoid against reactive oxygen species, produced during normal oxygen metabolism, or induced by exogenous damage, in addition, it possesses anti-inflammatory [12], a vasodilator [13] and angiogenic effects [14, 15]. The current study was carried out to assess the synergistic effect of both curcumin and quercetin on gamma-radiation-induced disorders in rats.

Ethics approval and consent to participate

The study protocol was reviewed and approved by the Institutional Review Board of the National Center for Radiation Research and Technology, Research Ethics Committee, REC-NCRRT (approval no. 45 A/21), Chair of the committee, Prof. Dr. Mahmoud M. Ahmed. All animals were treated in accordance with ARRIVE guidelines and regulations. All methods were performed in accordance with the relevant guidelines and regulations.

Experimental animals

A total number of 42 male Sprague Dawley albino rats of about 120 - 150 g body weight was used in this study. They were purchased from the animal Breeding House of the National Center for Radiation Research and Technology (NCRRT), Nasr City, Cairo, Egypt. Animals were housed in standard metal cages and maintained in conditions of good ventilation, normal temperatures, and humidity ranges and kept under observation for one week before experimentation. Rats were fed on standard pellets, containing all nutritive elements. Drinking water and food were provided ad libitum throughout the study.

Radiation facility

Irradiation was conducted at the National Center for Radiation Research and Technology (NCRRT), Nasr City, Cairo, Egypt. A Gamma Cell-40 (Cesium 137) was employed, and the dose rate was 0.61 Gy/minute during the experimental periods. Male rats were whole-body exposed to 8 Gy delivered as a fractionated dose (2 Gy every three days).

Preparation of Quercetin and Curcumin

Quercetin (Sigma Chemical Co., St. Louis, MO) dissolved in 1 ml of normal saline, administered orally by gavage after fasting overnight at doses (1.25 g/kg) body weight [16], and curcumin (Sigma Chemical Co., St. Louis, MO) was administered orally by gavage after fasting overnight at doses (100 mg/kg) body weight [17].

Experimental Design

In the present study, male albino rats weighing 120-150 g were divided into six groups: 7 rats each. Group, I was treated with saline and served as the control for 51 days. Group II received an oral daily dose of quercetin (1.25 g/kg) for successive 28 days and hung up for 9 days then continued administration again for 21 days. Group III received an oral daily dose of curcumin (100 mg/kg) at the same interval time as Group II. Groups IV, V and VI were exposed to a whole body of γ-irradiation started on day no 28 of the experiment at 2 Gy/72 hours (4 times up to 8 Gy). Group V received a combination treatment of quercetin and curcumin (1.25 g/kg), 100 mg/kg, respectively) at the same interval time as Group II. Group VI doesn’t receive any treatment till the end of the irradiation protocol (2 Gy/72 hours (4 times up to 8 Gy) and was treated post-irradiation with the same dose of quercetin and curcumin for 21 days. Seven rats from each group sacrificed 51 days of the total time of the experiment, as shown in Table 1.

Table 1 shows the experimental design and different treatments for the groups under study.

Groups	Days 1-28	Days 28- 37				Days 37-51 (14 days Post-Irradiation)
Groups	Days 1-28	Day 28	Day 31	Day 34	Day 35	Days 37-51 (14 days Post-Irradiation)
Group I	Saline 0.2ml orally	Hanged Up without any treatment for 9 days				Saline 0.2ml orally
Group II	Quercetin 1.25 g/kg/day					Quercetin 1.25 g/kg/day
GroupIII	Curcumin 100mg/kg/day					Curcumin 100mg/kg/day
Group IV		2Gy	2Gy	2Gy	2Gy
GroupV	Quercetin&Curcumin	2Gy	2Gy	2Gy	2Gy	Quercetin&Curcumin
group VI		2Gy	2Gy	2Gy	2Gy	Quercetin&Curcumin

Preparation of Samples

At the end of the study, the animals were sacrificed after anesthesia using thiopental [18]. Blood samples were withdrawn from each rat by heart puncture using the sterilized syringe, the blood was placed on ethylene diamine tetra acetic acid (EDTA) into tubes for hematological analysis and part of the samples was collected into heparin-treated tubes. Plasma samples were obtained by centrifugation at 3000 rpm for 10 min.

Hematology Analysis

Assessment of hematology profile

All hematological parameters: Hematological indicators including erythrocyte (RBC), leukocyte (WBCs) and platelets count (Plt), hemoglobin concentration (Hb), hematocrit (Hct %), mean erythrocyte hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and mean erythrocyte volume (MCV) were measured by using Automated Hematology Analyzer-Model MEK-6420K, Nihon Kohden Co. Japan [19].

Biochemical parameters

The extent of lipid peroxidation was assayed by the measurement of thiobarbituric acid reactive substances (TBARS) according to [20]. Blood-reduced glutathione (GSH) content was determined according to the method of [21]. AST & ALT activity was determined using the method described by [22]. Serum alkaline phosphatase (ALP) activity was measured according to [23]. The lipid profile tests including triglycerides, total cholesterol, high-density lipoprotein-cholesterol content (HDL-c), and low-density lipoprotein (LDL) content were evaluated using the method described by [24-26] using a UV/VIS T60 UV/VIS spectrophotometer.

Histological Methods

Small liver samples were fixed in neutral buffered formalin for 48 hours, embedded in paraffin after dehydration, cut into 5 μm sections and stained with hematoxylin and eosin (HE) staining [27] for the assessment of histopathological changes.

Statistical Analysis

The Statistical Package for the Social Sciences (SPSS/PC) computer program was used for statistical analysis of the results. Data were analyzed using one-way analysis of variance (ANOVA). The data were expressed as mean ± standard deviation (SD). Differences were considered significant at P ≤ 0.05.

FTIR spectroscopy

Fourier transform infrared spectra of liver samples was detected on a Jasco FT/IR 460 Plus (Japan) spectrometer. KBr sandwiches were pellet perfectly and all samples were prepared by mixing with them separately. All samples were recorded in the middle infrared frequency range in the spectral range from 400 - 4,000 cm^-1 with a speed of 2 mm/s at a resolution of 4 cm^-1 at room temperature. The bandwidth was measured at 50 % of the height of each peak. All procedures were done at the microanalytical center, Cairo University, Egypt.

Hematological Results

The results of Hb, RBCs, Hct %, MCH, MCHC, MCV, WBCs and platelet count (Plt) showed no statistical difference in their mean values following oral administration of quercetin or curcumin for 51 days as compared to the control group. Exposure of rats to whole-body gamma-irradiation at fractionated doses (2 Gy, four times, every three days) up to 8 Gy triggered a highly significant statistical decrease in the Hb, RBCs, MCH, MCHC, WBCs count, and platelet count (P < 0.01) with a significant statistical decrease in the Hct %, MCV on the 14^th day following irradiation process as compared to the control values (P < 0.05). The dual oral administration of both quercetin and curcumin pre-irradiation induced a highly significant increase in all studied parameters (P < 0.01) throughout the experimental times as compared to the corresponding irradiated group value. The co-administration of both quercetin and curcumin post-irradiation showed a highly significant increase in the RBCs count, WBCs, and platelets count on the 14^th day (P < 0.01) as compared to the irradiated group values indicating that administration of both quercetin and curcumin before exposure to gamma radiation was more effective than their post-irradiation administration. All the hematological parameters are listed in Table 2 and illustrated in Fig. 1.

Table 2 Hematological parameters Hb, RBCs, Hct %, MCH, MCHC, MCV, WBCs, and Plt for the six rat groups

Animal Group	Hb (g/dL)	RBCs 10⁶/µl	Hct %	MCH(pg)Hb/RBC	MCHC (g/dL) Hb/Hct	MCV (fL) Hct/RBC	WBCs c	Plt10³/µL
Control	15.9±0.29	5.5±0.29	43.5±0.19	31.95±0.14	38.79±0.19	79.17±0.19	9.59±0.19	341±1.19
Quercetin	15.6±0.23	5.0±0.30	42.1±0.33	31.35±0.13	38.06±0.33	76.62±0.33	9.30±0.17	325±1.33
% of change from control	-1.88%	-9.09%	-3.21%	-1.87%	-1.88%	-3.22%	-3.02%	-4.69%
P vs Control	(P> 0.05)	(P> 0.05)	(P> 0.05)	(P> 0.05)	(P>0.05)	(P> 0.05)	(P> 0.05)	(P> 0.05)
curcumin	15.2±0.24	5.2±0.45	41.4±0.24	30.55±0.22	37.08±0.42	75.34±0.42	9.32±0.18	327±1.42
% of change from control	-4.40%	-5.45%	-4.82%	-4.38%	-4.40%	-4.83%	-2.81%	-3.83%
P vs Control	(P> 0.05)	(P> 0.05)	(P> 0.05)	(P> 0.05)	(P> 0.05)	(P> 0.05)	(P> 0.05)	(P> 0.05)
Radiation	9.4±0.16	3.4±0.09	32.6±0.13	18.89±0.26	22.93±1.1	*59.33±1.4*	4.17±0.21	215±1.5
% of change from control	-40.88%	-38.18%	-25.05%	-40.87%	-40.88%	*-25.05%*	-56.51%	-36.95%
P vs Control	( P < 0.01)	( P < 0.01)	(P < 0.05)	(P <0.01)	(P< 0.01)	*(P < 0.05)*	(P < 0.01)	(P < 0.01)
Quercetin + curcumin +Radiation	13.9±0.5	4.9±0.22	43.5±0.14	27.93±0.34	33.91±1.08	80.17±1.5	7.86±0.13	319±2.2
% of change from radiation	47.87%	44.11%	33.43%	47.85%	47.88%	35.12%	88.48%	48.37%
P vs radiation	( P < 0.01)	( P < 0.01)	(P < 0.01)	(P <0.01)	(P< 0.01)	(P < 0.01)	( P < 0.01)	(P < 0.01)
Radiation + quercetin + curcumin	11.9 ±0.04	4.6±0.13	37.8±0. 22	23.91±0.38	29.03±1.3	66.79±1.4	6.05±0.10	297±1.64
% of change from radiation	26.59%	35.29%	15.95%	26.57%	26.60%	12.57%	45.08%	38.13%
P vs radiation	(P < 0.05)	( P < 0.01)	(P> 0.05)	(P < 0.05)	(P< 0.05)	(P> 0.05)	(P < 0.01)	(P < 0.01)

Biochemical Results

Plasma Thiobarbituric acid reactive substances (TBARS) concentration

The oral administration of Quer or Cur for consecutive 51 days showed no significant differences in plasma TBARS concentration as compared to the control untreated group. Exposure of rats to the fractionated doses of -irradiation at up to 8 Gy resulted in highly significant increases in the TBARS concentration (P < 0.01) as compared to the control values. Administration of both Quer and Cur before irradiation showed a significant decrease in the TBARS concentration (P < 0.05) as compared to the corresponding irradiated group, while their post-irradiation administration showed a non-significant decrease (P > 0.05) as compared to the corresponding irradiated group, the recorded values are shown graphically in Fig. 2 and listed in Table 3.

Table 3 Biochemical parameters, TBARS and GSH recorded for the different rat groups.

Animal Group

TBARS (nmol/ml)

GSH mg/dl

Control

105.24±3.19

25.42±1.13

Quercetin

% of changes from control

P vs Control

106.69±3.40

1.37 %

(P > 0.05)

26.59±1.28

4.60 %

(P > 0.05)

Curcumin

% of changes from control

P vs Control

107.30±3.47

1.95%

(P > 0.05)

24.97±1.04

- 1.77%

(P > 0.05)

Radiation

% of changes from control

P vs Control

163.10±3.61

54.97 %

(P < 0.01)

20.51±0.95

-19.31%

(P > 0.05)

Quercetin + curcumin +Radiation

% of change from radiation

P vs radiation

116.5±2.57

- 28.57%

(P < 0.05)

14.28±0.78

-30.37%

(P < 0.01)

Radiation + quercetin + curcumin

% of change from radiation

P vs radiation

145.03±3.38

- 11.07 %

(P > 0.05)

15.57±0.88

-24.08%

(P < 0.05)

Blood glutathione (GSH) content

The oral administration of Quer or Cur for consecutive 51 days showed no significant differences in blood GSH content as compared to the control untreated group. Exposure of rats to the fractionated doses of -irradiation at up to 8 Gy resulted in a non-significant statistical decrease in the mean value of serum GSH level(P > 0.05) as compared to the control group. Administration of both Quer and Cur before irradiation induced a highly significant decrease in the GSH level (P < 0.01), while their post-irradiation administration showed a significant statistical decrease in its level (P < 0.05) as compared to the corresponding irradiated group, the recorded values are displayed in Fig. 2 and Table 3.

Liver function Results

As presented in Table 4, and depicted in Fig. 3, the results showed that oral supplementation of Quer or Cur for consecutive 51 days did not cause a significant statistical difference in the serum ALT, AST, ALP activities and serum total protein content as compared to the control group values. A high significant statistical increase was noticed in the ALT, AST, and ALP activities on the 14^th day (P < 0.01) in the -irradiated rat group with a significant decrease in the serum total protein values (P < 0.05) as compared to the control group values. Pre-irradiation administration of both Quer and Cur resulted in a significant decrease in the serum ALT activity (P < 0.05) with a highly significant statistical increase (P < 0.01) in the total protein content, otherwise, there is a non-significant decrease in both AST and ALP activities (P > 0.05) as compared to the irradiated group recorded values. Oral administration of both Quer and Cur post-irradiation resulted in a non-significant amelioration of the radiation-induced decrease of ALT, AST and ALP activities values (P > 0.05) with an insignificant increase in the total protein content (P > 0.05) as compared to the corresponding irradiated group values.

Table 4 Liver function parameters recorded for the different rat groups.

Animal Groups	ALT (U/L)	AST (U/L)	*ALP(U/L)10**	T. Protein (g/l)
Control	29.46±0.81	38.47±1.05	17.918±0.307	7.06±0.11
Quercetin	30.9±0.92	39.18±0.97	17.827±0.299	7.12±0.09
% of changes from control	4.88%	1.84%	-0.50%	0.84%
P vs Control	(P> 0.05)	(P> 0.05)	(P> 0.05)	(P> 0.05)
Curcumin	29.94±0.84	38.64±1.04	17.868±0.281	7.13±0.10
% of changes from control	1.62%	0.44%	-0.27%	0.99%
P vs Control	(P> 0.05)	(P> 0.05)	(P> 0.05)	(P> 0.05)
Radiation	40.06±1.04	58.39±1.71	26.760±0.352	5.48±0.052
% of changes from control	39.37%	51.78%	49.34%	-22.37%
P vs Control	(P< 0.01)	(P< 0.01)	(P< 0.01)	(P < 0.05)
Quercetin + curcumin +Radiation	30.89±0.77	47.68±0.83	21.94±0.337	7.41±0.24
% of change from radiation	-22.89%	-18.34%	-18.01%	35.21%
P vs radiation	(P< 0.05)	(P> 0.05)	(P> 0.05)	(P < 0.01)
Radiation + quercetin + curcumin	35.17±1.06	51.43±0.96	25.831±0.345	6.33±0.13
% of change from radiation	-12.20%	-11.91%	-3.47%	15.51%
P vs radiation	(P> 0.05)	(P> 0.05)	(P> 0.05)	(P> 0.05)

Lipid profile Results

No significant differences were detected in the serum total cholesterol, triglycerides, HDL and LDL levels between the control group and the groups treated with quercetin or curcumin. The current experiment elucidated a highly significant elevation (P < 0.01) in the serum total cholesterol, triglycerides, HDL, and LDL concentrations following fractionated doses of g-radiation as compared to those of the control rats. Pre-irradiation treatment of rats with both Quer and Cur induced a significant decrease in the cholesterol level (P < 0.05) with a highly significant decrease in both triglycerides and HDL levels (P < 0.01) and a non-significant decrease in the LDL level (P > 0.05) as compared to the irradiated group values, whereas, its post-irradiation treatment exerted a non-significant decrease in the serum total cholesterol, triglycerides, and LDL (P > 0.05) levels with a significant decrease in the HDL level (P < 0.05) as compared to the irradiated group values (Table 5). A graphical illustration of the different lipid parameters recorded is shown in Fig. 4.

Table 5 Lipid profile parameters recorded for the different rat groups.

Animal Group	Cholesterol (mg/dl)	Triglycerides (mg/dl)	HDL (mg/dl.)	LDL (mg/dl)
Control	133.26±2.27	198.47±3.54	35.61±1.14	57.96±2.14
Quercetin	133.86±2.31	196.65±3.03	36.66±1.13	57.87±1.70
% of changes from control	0.45%	-0.91%	2.94%	-0.15%
P vs Control	(P> 0.05)	(P> 0.05)	(P>0.05)	(P>0.05)
Curcumin	133.61±2.17	196.18±3.14	37.47±1.29	56.91±1.19
% of changes from control	0.26%	-1.15%	5.22%	-1.81%
P vs Control	(P> 0.05)	(P> 0.05)	(P>0.05)	(P>0.05)
Radiation	188.40 ±2.29	318.32 ±4.66	48.28 ±1.40	76.46 ±2.07
% of changes from control	41.37%	60.38%	35.57%	31.91%
P vs Control	(P < 0.01)	(P < 0.01)	(P<0.01)	(P<0.01)
Quercetin + curcumin +Radiation	144.14 ±2.13	224.14 ±3.25	32.64 ±1.15	66.68 ±2.15
% of change from radiation	-23.49%	-29.58%	-32.39%	-12.79%
P vs radiation	(P < 0.05)	(P < 0.05)	(P<0.01)	(P>0.05
Radiation + quercetin + curcumin	171.42 ±2.18	281.55 ±3.19	34.47 ±1.19	80.64 ±2.39
% of change from radiation	-9.01%	-11.55%	-28.60%	5.46%
P vs radiation	(P> 0.05)	(P> 0.05)	(P<0.05)	(P>0.05)

Histopathological Results

Histological examination of a liver section of the control group revealed a normal histological appearance of the hepatocytes which are polygonal in shape and radially disposed of in the liver lobule. Each hepatic cell has a centrally located nucleus with one or two prominent nuclei. Occasionally, the liver cells appear binucleated. The spaces between the hepatic plates contain the liver sinusoids with phagocytic cells of the mononuclear phagocyte series known as Kupffer cells. Each hepatic lobule has a central vein at its core (Fig. 5, a). Liver sections of rats of the quercetin group showed the same normal histological appearance including a normal central vein, dilated portal vein, and bile duct, hepatocytes appeared with central vesicular nuclei where some hepatocytes are double nucleated as a sign of regeneration. Kupffer cells appeared activated as seen in (Fig. 5, b). Curcumin-treated rats showed the same normal architecture of hepatic parenchymal cells with the blood sinusoids that appeared occupied by blood cells with activated Kupffer cells. The portal tract appeared normally formed of the portal vein and bile duct (Fig. 5, c).

Whole-body exposure of rats of the current experiment to 8 Gy gamma irradiation delivered as a fractionated dose (2 Gy every 3 days) showed loss of the normal hepatic architectures with dilated central vein with corrugated walls and widened blood sinusoids. Some hepatocytes appeared degenerated with pyknotic nuclei and vacuolated cytoplasm (Fig. 5, d).

Administration of both quercetin and curcumin before gamma radiation exposure showed more or less normal hepatic architecture with the normal portal vein and bile duct. Hepatocytes appeared healthy with central vesicular nuclei, some of which appeared binucleated as a sign of regeneration as depicted in (Fig. 5, e). Administration of both quercetin and curcumin following gamma radiation exposure showed signs of recovery and tissue repair indicated by the well-developed hepatic architecture with a normal portal tract formed of the portal vein and bile duct, and widened blood sinusoids were still detected. Most of the hepatocytes appeared with central vesicular nuclei while, others have pyknotic nuclei (Fig. 5, f).

FTIR Spectroscopy

The average FTIR spectra of control liver tissues in 4000 to 400 cm⁻¹ regions is shown in Fig. 6. The main bands are labeled in the figure, and the band assignments are given in Table 6, [28-33].

Table 6 Band assignments of major transmissions in IR spectra of control liver tissue in 4000 to 400 cm⁻¹ regions.

Peak No	Wavenumber (cm^-1)	Definition of the spectral assignment [28-33]
1	3741	OH stretching
2	3289	Amide A: mainly N-H stretching of proteins
3	3006	Olefinic =CH stretching vibration: unsaturated lipids, cholesterol esters
4	2924	CH2 antisymmetric stretch: mainly lipids
5	2854	CH2 symmetric stretch: mainly lipids
6	1745	Saturated easter C=O stretch: Phospholipids, cholesterol easters
7	1651	Amide \|: Protein (80% C=O stretching, 10% N-H bending, 10% C-N stretching
8	1539	Amide II: Protein (60% N-H bending, 40% C-N stretching
9	1461	CH2 bending: mainly lipids, protein
10	1379	COO-symmetric stretch: fatty acids and amino acids
11	1237	PO2antisymmetric stretch: nucleic acids, phospholipids
12	1164	CO-O-C antisymmetric stretching: glycogen and nucleic acids
13	1097	PO2-symmetric stretch: nucleic acids, phospholipids

The average FTIR spectra of control, Quer, Cur, irradiated and combined Quer-Cur before and after irradiation-treated rat liver tissues in 4000 to 400 cm⁻¹ region is shown in Fig. 7. The figure reveals prominent differences between the average spectra belonging to the different groups. Subtle changes in a band shape, band position, and band intensity of vibrational bands represent changes in biomolecules' concentration, composition, and structure. It was observed that the broad peak of the OH group, CH₂, C=O, amide I, and C=C, respectively, had increased in intensity by varying the dopant material. All peaks before 1600 cm^-1 decreased in intensity by varying dopants. Peaks after 1600 cm^-1 increased in intensity by adding quercetin and decreased by adding curcumin. By adding quercetin, the peak positions were shifted to a lower wavenumber, while by adding curcumin, there were some fluctuations (many peaks increase in wavenumber and the others decrease). The reduced wavenumber may be due to the dopant material not interacting properly with the liver’s protein. The contrary is true; the increase in wavenumber is caused by the strong interaction between proteins and dopant material through the formation of hydrogen bonds.

The radiation effect on liver tissues was indicated by the shift of 3289 cm^-1 of NH stretching protein amide A to a higher intensity concerning the control and the disappearance of the OH stretching. It also shows a high decrease of the peaks 1745, 1651, 1539, and 1461 cm^-1 of phospholipids, amide 1 and amide 2 and lipid-protein, respectively, to a lower intensity concerning the control. There is also a shift in the peaks of 3006 cm^-1 for olefinic CH stretching for lipid and cholesterol, 2924 cm^-1 CH₂ for antisymmetric lipids, and a peak of 2854 cm^-1 for CH₂ symmetric lipids to lower intensity indicating the direct effect of radiation on liver tissues. It is shown from the figure that the radiation effect of the post-treated quercetin-curcumin group showed a decrease in the intensity of all peaks indicating its effect against radiation effect. There was a higher shift to higher intensity values for the irradiated pretreated quercetin-curcumin group in a close match with the control group. The combined doping of both quercetin and curcumin before and after irradiation showed a more significant effect in ameliorating the radiation effect on liver tissues, nearly restoring all the peaks to the control of the unirradiated one.

Due to the widespread usage of radiation in diagnosis, therapy, and industry; pharmacological intervention could be the most potent strategy to counter or ameliorate the injurious effects of radiation exposure [34]. The histological examination results included in the current study revealed that fractionated dose 8 Gy of γ-irradiation induced different histopathological changes indicated by loss of the normal hepatic architecture began with dilatation of the central vein and hepatic blood sinusoids, vacuolation and degeneration of its cytoplasm, pyknosis, and activation of Kupffer cells. The current results run in parallel with those reported earlier [35, 36].

Such alterations could be attributed to ionizing radiation which produces damaging cellular effects through water radiolysis, resulting in releasing of reactive oxygen species (ROS) in cells and the reduction of cellular antioxidants including GSH and enzymatic antioxidants. ROS can induce inflammatory reactions [37]. Radiation damage to living tissues was reported before to be a result of the overproduction of ROS [38]. Its overproduction increases lipid peroxidation in living cells, which increases oxidative stress enhanced by the disturbances of oxidants/antioxidants damage of the biological macromolecules including lipids, carbohydrates, proteins and nucleic acids, resulting in disturbance of the cellular homeostasis and the production of other reactive oxygen molecules that initiate more oxidative damage [39]. The results showed also that administration of both quercetin and curcumin antioxidants before and after radiation exposure decreased liver damage, however, the deleterious effects of Gamma-radiation were reduced more efficiently by its pre-irradiation administration which is confirmed by restoring all the peaks of the FTIR spectrum to be matching with the control unirradiated group.

Data from the present experiment showed that synergistic treatment of gamma-irradiated rats with both quercetin and curcumin antioxidants appears susceptible to lowering the statistical increase recorded in the ALT, AST, ALP, total cholesterol, triglycerides, HDL and LDL concentrations. The radioprotective efficiency of both antioxidants could be attributed to their role in scavenging free radicals [40]. Quercetin and curcumin are capable of alleviating intracellular oxidative stress [41]. Curcumin exerts various biological functions including antioxidation and anti-inflammation, and quercetin also has an important role in suppressing the expression of oxidative stress and inflammatory markers [42]. It could be concluded that quercetin and curcumin may exert a beneficial impact on gamma-irradiation-induced liver injury in rats.

Acknowledgements

The authors wish to acknowledge the National Centre for Radiation Research and Technology for their support during the experimental work.

Author contributions

S.A.A. corresponding author, supervision, designed, and organized the study. R.M.A. performed the experiments and wrote the original draft of the manuscript. S.M.S. contributed to the conduct of the study and supervision. I.Y.A. Performed the experiments and analyzed the data. W.M.K. Organized and critiqued the output for intellectual content. A.M.A. analyzed the data and drafted the manuscript. All authors discussed the results, reviewing and editing the manuscript.

Competing interests

The authors declare no competing interests.

Funding

Not Applicable.

Ethics approval and consent to participate.

Approval for experimental studies from National Center for Radiation Research and Technology, Research Ethics Committee, REC-NCRRT, Serial number of the protocol: 45 A/21

Consent for publication

All authors have read the submitted manuscript and consent for publication.

Availability of data and materials

All data are available from the corresponding author upon request.

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No competing interests reported.

Studies on the Effect of Curcumin and Quercetin in the Liver of Male Albino Rats Exposed to Gamma Irradiation

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Abstract

Figures

Introduction

Material and methods

Ethics approval and consent to participate

Results

Discussion

Conclusion

Declarations

References

Additional Declarations

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Version 1