Protective Effects And Its Relative Mechanisms Of Low Dose Ionizing Radiation On Diabetic Male Rat's Testis Damage

Diabetes mellitus (DM) is a chronic metabolic disorder which brings great danger to human health. Recent years, more and more people have focused on its reproductive system complication with incidence increase of diabetes mellitus and decrease of developing age. Many studies showed that the capacity of diabetic body to produce germs weakened and the percentage of immature and apoptotic germs increased. Testes were injured by reactive oxygen species (ROS) produced by high glucose and SOD, CAT and GSH-Px activities decreased and lipid peroxidation products increased, which resulted in sex and reproductive ability disorder of diabetes mellitus. It has profound significance to study the reproduction complication and its prevention of diabetes mellitus. Recent studies show that its main pathogenic mechanism is oxidative damage induced by hyperglycemia. ROS was produced by high glucose through many mechanisms (glucose oxidized and polyol stimulated), and then mitochondria was damaged and cytochrome c was released from it, and caspase-3 was activated and consequently lead to germ cells apoptosis. The resultant oxidative stress took critical effects in diabetic mellitus pathogenesis. Therefore, more and more scholars focused on the treatment methods with antioxidants.In the past twenty years, exciting effects induced by low dose radiation was studied extensively. LDR could stimulate DNA, RNA and protein synthesis, improve DNA repair, increase cell antioxidative ability, prolong cell survival time and activate body immune function. Pretreatment with nonlethal low-dose radiation has been shown to have protective effects against oxidative injury in animal tissues in which low dose irradiation increased endogenous antioxidants in animal tissues such as SOD, glutathione (GSH), glutathione peroxidase (GSH-Px), glutathione reductase (GR), catalase (CAT) and thioredoxin (Trx) in liver, spleen, brain and testis. These antioxidants inhibited oxidative damage in these organs further. The past studies reported that a dose of 0.5 Gy radiation increased SOD activity of pancreas in non-diabetic mouse and inhibited the decrease of SOD activity in pancreas in diabetic mouse induced by alloxan (ALX) or non-obese diabetic mouse and decreased lipid peroxidation products in plasma and pancreas in diabetic mouse induced by ALX significantly. These studies showed that LDR could prevent spontaneous or induced diabetes mellitus by ALX through increasing antioxidative ability of diabetic body. There were few reports about the effects of low dose radiation on diabetic reproduction complication. So the present study explored the effects and its relative mechanism of low dose radiation on diabetic reproduction complications, which might establish the theoretical and experimental basis to apply the low dose radiation to clinics.Immune and molecular biological techniques were used in the present study to examine the effects and its relative mechanisms of low dose irradiation on diabetic mellitus rats. The results showed that low dose irradiation decreased the ROS and lipid peroxidation product contents in diabetic rats'germ cells and increased the antioxidant ability. At the same time, LDR improved the disintegration of mitochondria membrane potential and reduced the Ca²⁺ concentration within the cells and maintained the cell membrane permeability, which protected the germ cells from damage produced by diabetes mellitus high glucose. The study still showed that LDR prevented the germs from apoptosis through increasing the expression of Bcl-2 gene and decreasing the expression of Bax, Cyt c, Fas, FasL and caspase-3 genes. That is LDR exerted its protection effects through the mutual roles of the above mentioned factors. The present study offered the scientific basis of LDR treating and preventing the development of diabetes mellitus.1. Rat diabetes mellitus modelMale Wistar rats had free access to water and a standard laboratory diet under 12/12 h light/dark cycle. Diabetes mellitus was induced by a single intraperitoneally injection of streptozotocin (STZ, 50 mg/kg body weight) after fasting overnight. Only animals that exhibited a blood glucose level of 16.7 mmol/L or higher and urine glucose more than"+++"were regarded as diabetic rats after they were injected with STZ for two days. The diabetic rats showed obvious polydipsia, polyphagia, polyuria and weight loss. These diabetic rats were similar to 1 type diabetes mellitus. And the diabetic rats were irradiated with 25, 50 and 75 mGy (dose rate was 12.5 mGy/min) X-ray every other day for one month. The normal control animals were sham-irradiated. The rats were killed after they were raised for another 4 or 8 weeks. Some diabetic rats were dealt with NAC as the positive medicine group in the 8 week DM rats experiment. These rats were given NAC (150 mg/kg body weight) by intragastric administration every other day for 4 weeks. Then the rats were raised for another 4 weeks. All the rats were killed at the end of 8th and 12th week after the rats suffered with DM. Serum and testes were collected and stored at -70℃for further examination and other testes were made into single cell suspension for cell apoptosis indexes research.2. Effects of low dose radiation on the TS, FSH and LH contents in diabetic rats serum8 week DM experiment results showed that TS and LH contents in diabetic rats'serum were significantly lower than those in normal control rats (P < 0.01). While TS and LH contents in 25 mGy, 50 mGy and NAC group were higher than those in the diabetic rats obviously (P < 0.01). There's no significant difference between each group for FSH level. 12 week DM experiment results showed that TS, FSH and LH contents in DM serum were lower than those in DM group significantly (P < 0.01), while TS, FSH and LH contents in 25 mGy irradiation group were obviously higher than those in DM group (P < 0.05 and P < 0.01). TS and FSH contents in 50 mGy irradiation group were higher than those in DM group (P < 0.05 and P < 0.01). FSH contents in 75 mGy radiation group were higher than those in DM group (P < 0.05). That is LDR increased androgen levels and prevented the diabetic rat reproduction complication through adjusting the roles of hypothalamus -pituitary gland-sexual gland axis.3. Effects of low dose radiation on the lipid peroxidation contents and antioxidative enzymes activities in diabetic rats serum and testes 8 week and 12 week DM experiment results all showed that MDA content in serum and testis and ROS level in diabetic rat germ cells increased significantly compared with the normal control group (P < 0.05 and P < 0.01), while MDA content in serum and testis and ROS level in LDR and NAC group were lower than those in DM group, especially in 25 and 50 mGy radiation group (P < 0.05 and P < 0.01). 12 week DM experiment results showed that 75 mGy radiation group decreased MDA level in serum and testis and ROS level in germ cells significantly compared with the DM group (P < 0.05 and P < 0.01). 8 week and 12 week DM experiment results all showed that antioxidative enzymes such as SOD and CAT activities decreased significantly compared with the normal control group (P < 0.05 and P < 0.01), while NAC, 50 and 75 mGy irradiation increased SOD activity in rats serum and testis (P < 0.05 and P < 0.01) and had no obvious effects on CAT activity in 8 week DM rats. Twelve week DM results showed that 25, 50 and 75 mGy radiation increased CAT activity in DM rat serum and testis and GSH level in serum significantly (P < 0.05 and P < 0.01), meanwhile SOD activity in three radiation group rats testes were higher than that in DM group (P < 0.05 and P < 0.01) and 50 mGy radiation increased SOD activity in serum significantly (P < 0.01) in. That's LDR cut down the oxidative stress damage induced by high glucose through improving the antioxidative ability and decreasing the peroxidized substances level in the diabetic rats.4. Effects of low dose radiation on NO contents, NOS and ATP enzymes activities in diabetic rats serum and testis8 week diabetic rats: NO content and NOS activity in serum and testis increased significantly compared with the normal control group (P < 0.01), while NO content in serum and testis and NOS activity in serum of NAC and LDR group were lower than those in DM group significantly (P < 0.05 and P < 0.01) and NOS activity in NAC, 25 and 50 mGy radiation group were lower than that in DM group (P < 0.05 and P < 0.01). 12 week diabetic rats: NO content in serum of DM group increased significantly compared with the normal control group (P < 0.01), while NO content in serum of 25 mGy irradiation group decreased obviously compared with the DM group (P < 0.01). NOS activity in diabetic rat serum increased a little and 50 mGy irradiation decreased NOS in serum significantly (P < 0.01). NO level and NOS activity in testis of DM rats were higher than those in the normal control rats (P < 0.05), while 25 mGy radiation decreased NO level and NOS activity in testis significantly (P < 0.05 and P < 0.01).Ca²⁺-ATP and Na+-K+-ATP enzymes activities in testis of 12 week diabetic rats were lower than those in control rats significantly (P < 0.05), while these two enzymes in the 50 and 75 mGy irradiated rats were higher than those in diabetic rats obviously (P < 0.05), and Ca²⁺-ATP enzyme in 25 mGy radiation group was higher than DM group (P < 0.05).5. Effects of low dose radiation on diabetic rats germ cell apoptosis8 week diabetic rats: Annexin V-PI kit was used to detect the apoptosis percentage in rat testis germ cells. Results showed that germ cells apoptosis percentage increased significantly than that in normal control rats (P < 0.01), while germ cells apoptosis percentage in NAC, 25, 50 and 75 mGy irradiation group decreased significantly (P < 0.05 and P < 0.01).12 week diabetic rats: Annexin V-PI kit and TUNEL kit were used to examine the germ cells apoptosis change. Results showed that germ cells apoptosis percentage in diabetic rats was obviously higher than that in control rats (P < 0.01), while germ cells apoptosis percentage in 25 and 50 mGy radiation group decreased significantly compared with that in DM group (P < 0.01). TUNEL positive cell nucleus showed brown yellow color and apoptotic germ cells mainly existed in spermatogonia and spermatocytes. TUNEL results showed that apoptosis germ cells in DM group were more than that in normal control rats and LDR decreased apoptotic germ cell number, which was consistent with the FCM results.6. Effects of low dose radiation on diabetic rats germ cell mitochondria membrane potential and Ca²⁺ expression percentageGerm cell mitochondria membrane potential in diabetic rats was obviously lower than that in control rats in 8 and 12 week diabetic rats with JC-1 and Rh 123 dyes respectively (P < 0.01), while mitochondria membrane potential in 50 and 75 mGy irradiation group and NAC group rats were higher than that in the diabetic rats significantly (P < 0.01). That is low dose radiation and NAC prevented germ cells mitochondria membrane potential from disintegration effectively.12 week diabetic rats: Ca²⁺ content in diabetic rats germ cells was higher than that in normal control rats (P < 0.01), while 50 mGy decreased and 75 mGy increased Ca²⁺ content in germ cells compared with the DM rats (P < 0.01). That is 50 and 75 mGy had contrary effects on germ cells Ca²⁺ level.7. Effects of low dose radiation on diabetic rats germ cell apoptosis relative proteins expression in diabetic ratsThe present experiment explored the change of some genes in death receptor and mitochondria way of apoptosis regulation. Protein expressions were examined with flow cytometer, immunohistochemistry and Western blot methods, and mRNA levels were examined with real-time RT-PCR methods. Fas, FasL, Cyt c, Bax and caspase-3 protein and their mRNA levels in diabetic rats were significantly higher and Bcl-2 gene level was obviously lower than those in control group (P < 0.05 and P < 0.01), while Fas, FasL, Cyt c, Bax and caspase-3 protein levels and their mRNA levels in irradiation group (25 and 50 mGy) were lower than and Bcl-2 gene expression level was higher than those in DM group (P < 0.05 and P < 0.01). That is low dose irradiation and NAC might prevent germ cells apoptosis by increasing anti-apoptosis proteins expression and decreasing the apoptosis induced protein expression.