Effects of Zingiber officinale hydro-alcoholic extract on HMG-COA reductase level in the testis of streptozotocin-induced diabetic rats

Introduction Cholesterol is a vital constituent of mammalian cell membranes, and comprises about 30% of all animal cell membranes, it maintains membranes and modulates membrane fluidity over the range of physiological temperatures, however, excess cellular and circulating cholesterol is harmful that can be related to some diseases, including coronary artery disease and atherosclerosis (1,2). The maintenance of cellular cholesterol homeostasis is precisely regulated by several feedback mechanisms. The major regulatory targets of these feedback mechanisms are HMG-CoA reductase in cholesterol biosynthesis pathway, the low-density lipoprotein (LDL) receptor in cholesterol uptake, and cholesterol 7α-hydroxylase in cholesterol http://www.herbmedpharmacol.com doi: 10.15171/jhp.2018.16


Introduction
Cholesterol is a vital constituent of mammalian cell membranes, and comprises about 30% of all animal cell membranes, it maintains membranes and modulates membrane fluidity over the range of physiological temperatures, however, excess cellular and circulating cholesterol is harmful that can be related to some diseases, including coronary artery disease and atherosclerosis (1,2).The maintenance of cellular cholesterol homeostasis is precisely regulated by several feedback mechanisms.The major regulatory targets of these feedback mechanisms are HMG-CoA reductase in cholesterol biosynthesis pathway, the low-density lipoprotein (LDL) receptor in cholesterol uptake, and cholesterol 7α-hydroxylase in cholesterol catabolism pathway (3)(4)(5).Diabetes mellitus has been revealed to be associated with sexual dysfunction in both sexes.Previous studies have shown that several phytoingredients have a potential influence in the regulation of cholesterol homeostasis in animal models (6)(7)(8).One of these phyto-ingredients is Zingiber officinal Roscoe, that is traditionally widely-utilized spice in worldwide.Some properties such as anti-inflammatory, antioxidant and anti-diabetic properties have been reported for Z. officinal (9)(10)(11).Some studies have shown that the ethanol extract of Z. officinale alleviates plasma lipids in cholesterol fed hyperlipidemia rabbits (12,13) and in streptozotocin (STZ)-induced diabetic rats (14).Also, LDL oxidation in atherosclerotic mice was inhibited by ethanol extract of Z. officinale in some studies (15).In addition to the alcohol extract of ginger, the aqueous extract of Z. officinale has also been shown to reduce lipid profile in serum of normal rat (16).These reported effects of Z. officinale may be resulted from inhibition of cellular cholesterol biosynthesis (15) and excitation of cholesterol removal from the body (17,18).To the best of our knowledge, the effects of hydro-alcoholic extract of Z. officinale on the HMG-CoA reductase level in the testis of STZ-induced diabetic rats, which is the main aim of this study, have not yet been well assessed.

Animals
In this experimental study a total 16 male Wistar rats with the average weight of 200-250 g were assessed.The rats were purchased from Research Center and Experimental Animal House of Ahvaz Jundishapur University of Medical Sciences (Ahvaz, Iran).Upon entrance, all the animals were accommodated in a well-ventilated room with a comparative temperature of 22 ± 2˚C and photoperiod of 12-hour light-dark cycle.During the experiment, all animals were given free access to standard food pellet and tap water.

Experimental design
For induction of diabetes, after one-week adaptation and following an overnight fasting, the rats were given a single intraperitoneal (ip) injection of STZ (Purchase from Sigma-Aldrich, USA) 60 mg/kg that was freshly dissolved in 0.1 M cold citrate buffer in pH 4.5.Also, the healthy control rats were injected alone with the same volume of sodium citrate buffer solution.After three days of STZ induction, blood specimens were collected from the nicked tail-vein, and the blood glucose levels were measured.The blood glucose more than 350 mg/dL was considered as diabetes.Two weeks after STZ injection, oral gavage of Z. officinale extract was initiated and continued for a period of 8 weeks.The rats were randomly categorized into four experimental groups, each group consisted of four rats, and assessed as follows: Group 1: Healthy control was orally gavaged with 1.5 mL/ kg distilled water once daily.Group 2: Diabetic control was orally gavaged with 1.5 mL/ kg distilled water once daily.Group 3: Diabetic rats were orally gavaged with 200 mg/kg of the extract once daily.Group 4: Diabetic rats were orally gavaged with 400 mg/kg of the extract once daily.

Sample collection
The fasted rats in all groups at the end of eight-week treatment were weighed and then sacrificed after anesthetizing.By using not heparinized syringes, fresh blood samples were directly obtained via cardiac puncture and sera were separated by centrifugation for biochemical measurements.The testis was immediately isolated and washed with cold normal saline solution, then stored at -80˚C for later assays.
Preparation of Zingiber officinale hydro-alcoholic extract Dried roots of Z. officinale were purchased from a local company, Gol Darou, Isfahan, Iran.For extraction, 200 g of dry roots were grounded using an electric blender, and soaked in 1400 mL of 70% methanol solution (v/v) for 3 days.The extract was filtered through the Whatman filter paper, concentrated in a rotoevaporator, and dried with Freeze Dryer.The obtained extract, dry powder, was calculated 12.5%.

Western blot analysis
Western blot Analysis was performed on the supernatant fraction of homogenized rat's testis.By Bradford method, concentrations of total protein on the supernatant were measured.Seventy micrograms of protein were loaded onto 8% sodium dodecyl sulfate (SDS)-polyacrylamide gel for electrophoresis (SDS-PAGE).After SDS-PAGE, by BIO-RAD transfer system (USA), Protein blots transferred to a methanol pre-activated polyvinylidene difluoride (PVDF) membrane.After blotting, the membrane was blocked with skimmed milk 5% for 1.5 hours with gradually shakes.Subsequently, blots were probed with antibody specific for HMG-CoA reductase (1:5000 dilution) (Santa Cruz Biotechnology), and reprobed for β-actin (1:4000 dilution) (sc-130656; Santa Cruz Biotechnology) to serve as a loading control.Bound antibody was probed with secondary antibody, goat antirabbit IgG-HRP (horse radish peroxidase) (sc-2030; Santa Cruz) diluted 1:10 000 in 3% BSA (bovine serum albumin) for 1h at room temperature.After each step membranes were washed thrice with Tris-buffered saline-Tween (TBST) 0.1%.Finally, the blots were visualized with Chemi-Doc gel documentation system (Bio-Rad, Hercules, CA) using enhanced chemi luminescence (ECL) western blot detection kit according to the manufacturer's protocol.By using ImageJ software optical densities of bands were measured and quantified as ratio to β-actin.

Statistical analysis
All statistical analysis was carried out with SPSS software (SPSS Inc., Chicago, IL).Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey post hoc test.The comparison of data with normal distribution including blood glucose and body weight was done by ANOVA test.And, the data with no normal distribution were analyzed by Kruskal-Wallis test.The results were reported as means ± standard deviation (SD).A P value < 0.05 was considered statistically significant difference.

Results
Effects of Zingiber officinale extract on the biochemical parameters Before induction of diabetes, the mean of body weight was similar between groups and there was no significant difference in fasting blood glucose concentration.The effect of Z. officinale extract on the body weight, insulin level, and fasting blood glucose concentrations were assessed and the results are presented elsewhere (19).
Effects of Zingiber officinale extract on the rats' testis HMG-COA reductase level As illustrated in Figure 1, at the end of the experimental period in western blot analysis, a significant decrease in HMG-COA reductase protein level was observed in diabetic rats treated with 200 and 400 mg/kg of Z. officinale extract compared to controls (P < 0.01).No statistically significant difference for HMG-COA reductase protein level was observed between healthy controls and diabetic controls (P = 0.208).

Discussion
Hypercholesterolemia is common in diabetic patients and is a risk factor for coronary heart disease (CHD) according to reported by numerous studies (20)(21)(22).Some diabetic medications have troublesome side-effects, so there is increasing tendency to use of natural therapies.In traditional medicine, herbs are rich sources of therapeutic compounds for relieving symptoms of illness such as diabetes and hyperglycemia.Z. officinale is used as an important herbal remedy which has a reducing effect on blood glucose and cholesterol according to the results from previous studies (9-11,23) However, no studies have been done about the effects of Z. officinale hydro-alcoholic extract on the HMG-COA reductase expression in testis of diabetic rats.In the present study we have investigated this issue.Our results showed that in STZ-induced diabetic rats' body weight was significantly decreased compared to healthy control.Based on a study conducted by Kusari et al, in STZ-induced diabetes loss of body weight was observed.This reduction was related to loss of skeletal muscle resulting from hyperglycemia or high urine excretion and dehydration (24).Also, Eleazu et al showed that the reason for the loss of body weight was resulted from the degradation of muscle proteins similar to the results of our study (25).According to the present study, Z. officinale extract can prevent weight loss in rats with STZ-induced diabetic and this effect may be related to its hypoglycemic properties.Furthermore, this result is consistent with the findings reported in previous studies (26,27).
In the present study, serum insulin level was significantly reduced in diabetic control in comparison with healthy control, ones after 10 weeks of diabetes induction.It has been demonstrated the diabetogenic effect of STZ is resulted from the permanent destruction of the β-cells and deficiency of insulin secretion (28), so this finding can verify our result.On the other hand, serum insulin level was significantly higher in diabetic rats treated with 400 mg/kg of Z. officinale extract than diabetic controls (29,30).Z. officinale extract may be stimulating β cells to secrete insulin or releases this hormone of insulin-granule complex.This finding is in agreement with our results (29,30).In diabetic controls compared to healthy control ones, following decreased insulin secretion, the fasting blood glucose concentration was significantly increased.Some other animal studies have reported similar findings (11,28,29).We observed that decrease of fasting blood glucose in diabetic rats treated with Z. officinale extract was dose dependent which is in agreement with other findings reported by other studies (11,23,27).Hypoglycemic property of Z. officinal is probably related to certain plant components such as polyphenol constituents and flavonoids (9).Flavonoids may decrease plasma and hepatic cholesterol level due to the decrease hepatic HMG-CoA reductase level (31).Raw ginger extract can decrease serum cholesterol and triacylglycerol levels in diabetic rats and it has the great effect for controlling of diabetic complications (32).Our results indicated there was a significant reduction in HMG-CoA reductase level after treatment with both doses of Z. officinal extract.Cholesterol is a precursor to testicular androgens production in Leydig cells.It was assumed that decrease of total cholesterol level by reduction of HMG-CoA reductase with Z. officinal may create sexual hormone dysfunction and it is a side effect to use for diabetes disease.But Kamtchouing et al reported that the aqueous extract of Z. officinal significantly increases the serum testosterone level (33) that is in contrast with its cholesterol-lowering effect.The mechanism of action of ginger for increasing the level of testosterone is unknown.However, it has been suggested that ginger may stimulate the production of luteinizing hormone (LH) which in turn more stimulate Leydig cells to produce testosterone (34).The effect of Z. officinal roots on fertility in diabetic male rats has been reported previously.It can enhance the serum testosterone and improve the sperm motility (35).In a study ginger which was added to semen fluid could induce toxic effects on sperm motility and morphology.The result was dose and time-dependent, but in several studies, ginger showed useful effects on the semen parameters (36).

Conclusion
The oral administration of Z. officinale hydro-alcoholic extract has been shown to increases body weight, reduce blood glucose concentration and improve serum insulin levels in STZ-induced diabetic rats.However, the extract significantly decreased HMG-COA reductase level in diabetic rat's testis.In general, ginger shows effective glycaemic control properties, and, has a potential influence in the regulation of cholesterol homeostasis through modulation of HMG-COA reductase level in testis of diabetic rats.

Figure 1 Figure 1
Figure 1 Effect of Z. officinal extract at the end of 8 weeks intervention on the HMG-COA reductase expression.(A) Representative of immune-blots demonstrating specific bands for HMG-COA reductase (B) Graphical