Osteoporosis is the most recent disease are spread widely among women than men. This disease has bad effect not only on bone structure and physiology but also on different organs especially liver. So that, different medication have been used for treatment. Globally prevalent postmenopausal osteoporosis has a major negative impact on women's quality of life[1]. The variations in postmenopausal women with normal bone mineral density, osteoporosis, and osteoporotic fractures' health-associated quality of life are yet unknown[2]. 

Bone physiology is a balance between bonr formation by osteoblast and bone resorption by osteoclast. If osteoclast increases in number and activity more than osteoblasts, this will cause osteoporosis. This is done through reducing bone mineral density. Estrogen declines one of the reasons behind osteoporosis in pstmenupausal women[3,4]. 

Osteoporosis during menopause is associated with an estrogen deficit that arises from the ovaries ceasing to function as people age. The role of estrogen in the process of bone remodeling is now well understood[5]. Primary osteoporosis arises from the natural aging process in humans, while secondary osteoporosis is caused by particular systemic illnesses and clinical pathologies[6,7].

Medications for osteoporosis fall into one of three categories: anabolic (promoting bone growth), antiresorptive (blocking bone resorption), or both. Although all currently approved drugs lower the incidence of fragility fractures in high-risk individuals, most patients with established disease are typically not able to entirely repair bone strength due to these medications[8,9]. 

According to study, estrogen contributes to the production of new bone as well as the inhibition of bone resorption. As a result of ovariectomy, endocrine system has changed causing hypogonadism, then leading to the development and accelerating bone resorption (loss), followed by appearing osteoporosis risk[10].

In spite of the changes in the hormone post ovareictomy in rodents, estrogen  declines rapidly and permanently in comparsion with other animal models of estrogen hormone deficiency. The estrogen deficiency may be involved in VCD(4- vinylcyclohexene) chemical administration which induce ovary failure leading to lowering estrogen levels in addition to buserelin treatment. This is by suppressing hypothalamous pituitary ovarian axis [11] 

In the recent decades, there were several trails for natural oils as Hormone replacement Therapy(HRT). Omega-3 fatty acids is mainly found in Flaxseed oil Linum usitatissimum and has also been proved to reduce the incidence and effects of chronic inflammation and inflammatory bowel disease. However, direct evidence of dietary PUFA's potentially positive effects on human osteoporosis is still unavailable. Using omega-3 fatty acids lowers plasma triglycerides (TG) and small dense LP while rising large buoyant LDL and HDL-C, which can result in a better lipid profile [12]. Other trials have shown the role of omega-3 fatty acid supplementation in the treatment of dyslipidemia[13].

 The research of [14] imply that ALA in falxseed oil protects bones from LPS-induced inflammation and injury by blocking the proinflammatory molecules levels. As a result, synaptic longevity, reproductive success and memory have been  improved successively, in addition to differentiation of behavior improving mood, and sexuality [15]. Phytoestrogens have also been shown to have a pain-relieving effect in some trials [16]

Symptoms of low estrogen levels is irregular or missed periods, hot flushes, night sweats, insomnia, vaginal dryness, low libido, moodiness or irritability, headaches and dry skin [17]. Alterations and degradation of bone homeostasis and structure occur with postmenopausal osteoporosis [5,6]. This is caused by an imbalance between the activities of osteoblasts and osteoclasts [7]. Following menopause, an estrogen deficit usually accelerates bone turnover, with bone resorption overtaking bone production [8]. Many explanations have been put out, such as the function of aromatase in converting testosterone to estradiol and the possibility that a drop in serum aromatase levels could result in a female's lack of estradiol[8, 7].

Because phytoestrogens share structural similarities with the main female sex hormone, 17β-estradiol (E2), they may offer an alternative to anti-osteoporosis medications [10] 

Flaxseed (Linum usitatissimum) is known by itʼs active components that are necessary for health against liver dysfunction, cardiovascular disease, concerned obesity[18]. it is a perfect source of proteins, oils and diatery fibers nin addition to phytochemical antioxidant and lignan. It is also rich of omega-3 and omega-6[19].

One of these phytpestrogens is Soybeans. It is are a rich source of isoflavonoids, such as genistein and daidzein, which bind to E2 receptors (ERs). There is conflicting evidence about how soy isoflavonoid extract affects postmenopausal women's bone mineral density (BMD). While a different study found no favorable effect of soy isoflavone on spine BMD [20], a meta-analysis study found that soy isoflavone treatment leads in an increase in BMD in the lumbar spine but not in the femur neck, whole hip, or trochanter. Regular tea drinking has been linked to some favorable benefits on bone density; however, most studies are retrospective or cross-sectional in nature[21].

The liver is the most essential organ, as it regulates numerous physiological processes in the body. It performs a variety of critical tasks, including metabolism, secretion, and storage . as well as It has a large capacity to detoxicate hazardous chemicals and produce beneficial compounds fundamentals[22]. 

Results of previous researches showed that liver disease leads to osteoporosis[23]. Therefore, this study aimed to investigate whether osteoporosis leads to liver dysfunction and if flaxseed oil could improve liver state in ovarectomized rats in comparsion to estradiol, by examining histopathology and liver function markers.

Fifty Albino female rats purchased from the Animal Field of the College of Veterinary Medicine and randomly divided into five groups of ten in each group. These rats were (11±4) weeks old and weighed (175± 6) grams. Lived in a typical habitat with 12 hours of darkness and 12 hours of light, a standard food, and clean water. Under standard surgical circumstances, ketamine (10% IP; ketamine, alfasan, Holland) and xylazine were used (2 percent IP; alfasan, Holand). ovarectomy procedures were performed for G2, G3, G4, and G5 then remained for 30 days for ensuring the happening of osteoporosis. G1 was treated orally with maize oil, G2 was merely overiectomised, and G3 was both overiectomised and treated orally with (20 mg/k) flaxseed oil, G4 was overiectomized and injected intraperoteneally with (8mg/k)17-beta Estradiol Benzuet, whereas G5 was overiectomized and treated with both flaxseed oil and 17 -beta Estradiol Benzuet.

Preparation of Flaxseed Oil

Flaxseed oil capsules (Blackmore, Australia) were purchased, each containing 1gm of flaxseed oil, which was mixed with maize oil to obtain a final concentration of 20 mg/kg.BW. 

beta Estradiol Benzuet calculation dose

The veterinary 17-beta Estradiol Benzuet dosage was determined and utilized for 30 days at 8mg/kg B.W. IP.

Liver Functions Estimation

            After ending of the the experiment period of 60 days, blood samples were collected to estimating  liver function. This is done following the method of [24]. The serum samples were kept in refrigerator till testing time. Serum were used for estimating the values of liver enzymes(AST, ALT).

Histopathological examination of liver

            Liver tissues were preserved in a 10% buffered neutral paraformaldehyde solution.then having been processed and embedded in paraffin  The H&E stain was applied to thin paraffin slices (5 μm). light microscope with power 40X and 10 X was used for examination. After processing, fixed tissues were inserted into paraffin blocks. 4 µm thick sections were stained with hematoxylin and eosin. After that, acamera (Olympus DP72-USA) was used to take pictures and a microscope (OlympusBx-Ucb - USA) was used to inspect the liver(25)..

Statistical analysis

Statistical research entails the analysis of data provided as mean SD. Multiple comparisons were performed using one-way ANOVA, with the least significant difference (LSD) used as a post hoc test. The significance criteria was set at the 0.05 level of probability. All statistical analyses were performed using SPSS (Inc., Chicago, IL, USA) software version 258.          

Liver Functions

            Liver functions are investigated through estimating the values of two enzymes (AST) and(ALT) at the end of the treatment period. The results in table (1) showed that serum AST activity is increased significantly at (P≤0.05) with mean value (227 ±14.3)IU/liter and (246 ± 26.8)IU/liter in both negative and G3 groups respectively in comparison with positive control group, while G4 group decreased significantly at  (P≤0.05) but still higher than positive control with mean value (165 ± 25.1)IU/liter. G5 group is significantly posses the highest mean value of AST among all groups with value (1023 ± 25.5)IU/liter.

                Serum ALT enzyme activity is also estimated to detect the state of liver function in all groups. The results of table(1) showed a significant increase at (P≤0.05) for G2 , G3, G4 and G5 groups with means values (68.2 ± 10.2)IU/liter , (88.2 ± 7.9)IU/liter, (55.2 ± 7.6)IU/liter and (611.4 ± 12.7)IU/liter respectively in comparsion to positive control group (39.4 ± 5.02)IU/liter. the highest mean value is in the combination group G5 with mean value (611.4 ± 12.7)IU/liter.

Table (1): liver functions by means (AST and ALT) ((IU/liter) in all groups. 

*data as (mean ±SD). Means with different letters in same column are in significant difference at (P≤0.05).

Hepatic histopathological results

Figure(1):photomicrographs shows liver tissue of (G1) in comparsion to (G2) where: central vein(CV), with normal hexagonal hepatocytes(n). showed necrosis(  ), apoptosis(       )  , vaculation

(       ), inflammatory cells(      )(H&E X10 &X40)

The figure(1) shows the effect of osteoporosis disease on The liver in the group(G2) which has showed clear hydropic degeneration (vaculation)as a sort of liver damage with signs of hepatocytic necrosis and apoptosis (formation of apoptotic bodies) with the presence of inflammatory cells, in comparsion to G1 of liver in normal rats which appears hepatocytes in hexagonal shape arround central vein. Some of these cell were of binucleated state.

The result of liver histopathology shows through figure(2) of (G3) of osteoporotic rats treated with estradiol hormone that their liver have undergone regeneration by the formation of binucleated hepatocytes( containes two nuclei shows activity), in spite of mild degree of necrosis in the same section. there is also active rounded cantral basophilic nuclei. in comparation with the liver of osteoporotic rats without treatment(G2). 

On the other hand, in figure(3), (G4) of flaxseed oil group, it is noitced that there is regeneration by means of radially arrangement of hepatocytes inspite of rhe congestion of central vein and mild exsistance of bilirubin, As well as, most hepatocytes have binucleated, hexagonal and normal shape with mild hydropic degenaration. Also, it can be noitced that there is a case of radiation in hepatocytes around central vein(CV).

In spite of the effect of esradiol hormone and flaxseed oil when they were used separatly, towards regeneration. The combination of both of them in (G5) group has worse effect towards degeneration, as shown in figure(4). there were hard changes by means of vaculation of hepatocytes in addition to degeneration , congestion of bile ducts and sever necrosis, in comparsion to G1, G3 and G4.

Liver is the largest and most crucial organ in the body which plays an important roles by regulation of different physiological biochemical processes in the body like secretion, metabolism, and storage. Its function also involves detoxtification of poisioning and toxic compounds and substances in addition ti the synthesis of important and useful ones[26].

1. Liver Enzymes

Increasing levels of AST and ALT significantly indicates that there toxicity in the liver in addition to the possibility of hepatocytolysis only when transferase  enzymes increased to 100 times in comparsion to normal groups[27]. The results of increasing AST and ALT shoes that there is direct effect of flaxseed oil and estrogen+flaxseed oil treatment on liver functions leading to limiting hepatolysis[27] as the direct effect with OVX group. While there no effect of estrogen hormone on liver function just like positive control group.The result of the recent study comes to be nearly similar to the result of 

[28] who has a higher means value of AST when the experiment affected rats with hepatolipidemia and  are administrated with Flaxseed oil in 2 and 4 weeks then declined at 6 week lower than positive normal group, while ALT values declines significanly to be incompatable to the recent study results. The recent study has a significant increasing mean value in negative, FO, ES and EFO groups in comparion to positive normal group.

The recent study leaded to the result of AST and ALT values to be incombataple with the study of  [29] who refers that treated rats with flaxseed oil leading to significant decreasing in ALT and AST concentrations in serum. ALT and AST are used as markers for detecting the state liver of damage⁴. flaxseed oil didn’t have an effect on reducing  rising values of AST and ALT in G2.  This is an indication of liver dysfunction. In opposit to estradiol which reduced the higher values of both markers. It is thought that the incidence of necrosis in liver tissue, participate in releasing high amount of liver marker AST into blood stream [30]. the result of the present study didn’t compatable with study of [30].

In some o It is thought that omega-3 fatty acid in flaxseed oil has a mechanisim to protect liver from damage involving stabilization activity on hepatocytes membrane⁴. It is found through the study of [31].The fixed value of AST and ALT may due to the effect of FO or ES may preventing reduction of BALP serum levels which in turn does not change levels of transaminases serum, in similar way when [32] have used Aldornate therapy which preserved the reduction of BALP then don’t effect of transaminase⁶. 

According to the study of [33], the value of ALT and AST activities are reduced in murin which receives higher amount of carbohydrates intake, when they are administrated with omega-3 fatty acid as treatment. This similar to the finding , who find that ALT activity is decreased in rats with induction of diethylnitrosamine toxity and treated then with omega-3 FA. Increasing levels of AST and ALT, indicates to cellular breakdown, decreasing of functional integrity, or/and cellular perimeability [34]. 

FO may repair the damage of liver by two possible directions: the first way is the administration of FO leads to increasing SOD, GPx and catalase levels in liver leading to enhancing protective activity against ROS. Second way is the existence of omega-3 in FO may replace polyunsaturated fattyacids(PUFA) components in cellular membrane which is attacked by O2 free radicals [35]. Flaxseed oil is considered as a basic source of omega-3 which change fatty acid composition in membranes , so affect the function and structure. FO accelaerte repairing and regenerating injured organelles such as lysosomes, mitochondria and plasma membrane by means of activation mechanism of endogenous antioxidant defence[36]. A decrease in antioxidant activity is characterized by the accumulation of strongly reactive free radicals, which cause negative effects including loss of cell membrane survival and functions [37].

Changes in serum hepatic transaminase levels indicate liver necrosis and  damage. Many studies, reported liver transaminase dysfunction due to intracellular lipid aggregation and microvesicular steatosis [39]. Increased hepatic transaminases leave to the plasma from injured hepatic cells in hyperlipidemia. As a result, the increase in hepatic transaminase activities in plasma may be primarily due to leakage of these enzymes from the liver cytosol into the bloodstream [40]. This leakage causes a reduction in GOT and GPT levels in hepatic cells but an elevated serum levels [41].

[42] also show that When compared to the sham-operated control groups, the plasma level of ALT was substantially higher in both the 5wk and 10wk OVX rats (P<0.001 for both).  AST levels were almost the same as in control rats, but slightly lower than in the 5wks OVX population (P<0.001). The ALT level in the 5wks OVX rats was slightly lower relative to the only OVX category (P<0.001) that reached its value in the sham-operated samples. 

The results of the present study agrees with results of previos researchs when the researcher used flaxsed supplements which rises levels of ALT and AST in serum. 

1. Liver Histopathology

The present study aimed to study the protective effect of flaxseed oil on improving the damage of liver resulted from osteoporosis. This is done by estimating two of liver markers and histopathology in the organ tissue in ovarectomized rats.  Through researches on osteoporosis, it is noitced in women that osteoporosis always associated to liver dysfunction [44].

Through the present study , it is noticed that the reduction of estrogen caused also in damage of hepatocytes by increases levels of hepatocytes damage.The addition of flaxseed oil as IP injection or diet modifies the metabolism of fatty acids, hence changing the levels of pro-inflammatory mediators and atherogenic lipids, which are markers of the modulation of inflammatory illnesses. It has been demonstrated that flaxseed oil prolongs the liver of irradiated mice, indicating its potency as a preventative measure against the radiation-induced alterations that degrade the liver. Through the present study , It is thought that linoleic acid (LA) and Alpha-linolenic acid(ALA)of flaxseed oil participate to improve liver function and structure and this is agree with the study of [32] when used flaxseed used as supplement for wistar rats.

Flaxseed oil and lignan have been shown to have protective properties against hepatotoxicity and nephrotoxicity [19,20]. However, flaxseed oil's ability to reduce toxicity. It is shown through the present study that falxseed oil and estrogen hormone worked separtley to reduce the effect of osteporosis . this is agreed with study of [30] when he found that flaxssed ,that is used as pretreatment drug, improve liver  tissue state after poisioning rats with lead metal [30]. there is aclear appearance of normal nuclei in binucleated cellsand radical hexagonal hepatocytes.

Through the present study , the researcher can conclude that osteoporosis can cause damage in liver architecture, then elevating values of ALT and AST. The this is due to necrosis in hepaocytes causes releasing of high amounts of ALT and AST. 

Also, It can be concluded that flaxseed oil and 17β-estradiol can have a positive effect on damaged liver from osteoporosis by regenerated its tissues towards normality. Flaxssed oil had affecting on levels of ALT and AST by rising their levels in blood stream, either used singularly or synergestically with eatrdiol hormone. Whereas estradiol hormone alone had a reducing effect on AST and ALT.

The authors declare that they have no conflict of interest

No funding sources

The study was approved by the University of Al-Qadisiyah.

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