Anti-aging Effect Of Oviductus Ranae And Its Mechanism

Aging is begun and accelerated after reproduction maturity. It is characterized by cumulative, universal, progressive and endogenous vital process, which is the common in nature of all creatures. Organism has three vital process phases, growth and development phase, reproductive period and senescence phase. Different species have similar mechanism of duration of life decision. In 21st centuries, human society is faced with a severe challenge of aging of the population. In 2007, United Nations Economic and Social Affairs published the《2007 World Economic and Social Survey》. The report is showed that because of fertility decline and increased life expectancy, most countries in the world are rapidly entering the aging society. From 2005 to 2050 years, half of the world's population increasing is the old over 60.Population of the senior over 80 will increase from 90 to 400 million and many kind of senile diseases will be followed as well. Therefore, how to delay senility has been one of the hot spot of international scholars to research. It is thus clear that the research of aging and anti-aging field and exploration of anti-aging drugs is of vital importance to improve the quality of human life.Oviductus Ranae (OR) is the Chinese forest female frog's((Rana temporaria chensinesis David)) fallopian tubes, obtained by the collect and process drying. It has the ability of tonify the kidney and replenish essence, nourishing yin and moistening lung and is used in the symptom including yin asthenia and physically weak, lethargy, heart palpitations and insomnia, night sweat and coughing up blood. The ingredients mainly contain protein and amino acids, fatty acids (saturated and unsaturated), hormones, steroids, inorganic elements, vitamins and so on. Now, it had reported that OR was able to significant prolongation mean life span and maximum lifes pan of Drosophila. Its anti-aging mechanism was to heighten activity of SOD and to decrease level of LPO. OR had also many effects incuding anti-fatigue, to adjust nerve immunization and lipometbolism.In our previous study, we also found that OR could increase the activity of superoxide dismutase(SOD), inhibit free radical accumulation in the reproductive organs and resist the aging of cells and degeneration of tissues and significantly increased estrogen levels organs in aged mouse induced by D-galactose. It could increase the ovary index and the uterus index, ameliorate their pathological change to improve the decaying ovary and uterus. We had made the YiFuling Soft Gelatin Capsules (YFL) with OR as principal drug for Climacteric Syndrome. Animal experiments showed that YFL could significantly elevate estrogens content in vivo and significantly improve uterus index, adrenal gland index. YFL could improve the uterine morphological structures in pathological condition and increase the number and secretion of gland and make uterine wall thickening. YFL had already become a hospital pharmaceutics (licensed No. XZ20010003).YFL was applicated in clinical treatment for many years and found it had better treatment effectiveness. We had also developed Kun Li tablet with OR, Ginseng, Angelica as healthy food (register No. G20090379). Kun Li tablet had obviously effect of reducing chloasma. We also obtained Chines patent of invention (patent number ZL01107471.X).Free radical theory was the best important theory in numerous causes of senescence. Free radical theory was supported by abundant experimental evidences. Although others causes of senescence had individual strong point, only free radical theory could contact with others theories and became a center of causes of senescence. Free radical theory was one of persuasivest theories to be verificated by theory and experiments study in 50 years. In recently years, researchers showed that as a normal and complicated life process, senescence was involved in a series of routine changing in various tissues and cells. Many studies indicated that cellular senescence was implemented via some signaling transduction pathways which a lot of cytokines were involved in. The most classical cell senescence pathways were P161NK4a-Rb and p53-p21WAF1/ClP1-Rb. When the key regulating factors (p16, p21, cyclin, CDK and Rb, et al), in two pathways were changed, cellular senescence would be delayed. Now some scientists found inhibition of p21-mediated reactive oxygen species (ROS) accumulation could rescue p21-induced senescence. They also showed that the p16INK4A-Rb pathway cooperates with mitogenic signals to induce elevated intracellular levels of ROS in human senescent cells. Phospho-Rb and its regulating factors (cyclin and CDK) might be playing a significant role in the senescence process.References analysis of OR discovered mechinsm of anti-aging in OR was related to antioxidation. Therefore we thought while OR inhibited ROS accumulation to anti-aging, OR regulated key factors expression of the two cell aging pathways was another possible anti-aging role of OR. It was reported that correlation of antioxidant effect of OR with regulate key factors expression effect of OR. In this study, we were going to investigat the changes of some key regulators of cellular senescence signal transduction pathway p53-p21-Rb and pl6-Rb, including p21, p16, cyclinDl, cyclinD3, CDK6,pRb,and detect the activity level changes of superoxide dismutase (SOD),CuZnSOD, peroxidase(POD) and total antioxidation capacity(T-AOC) in rats serum and liver, and observe changes of pathology of liver, uterus, ovary after taking OR by using the sub-acutely aging model rats induced D-galactose (D-gal), so as to find the mechanism of OR in anti-aging and correlation of antioxidation and these key regulating factors,to observe effect of OR in female aged rats estrus cycle, to investigate the mechanism of OR in delaying reproductive senescence with molecular biology techniques. This study could provide theoretical and experimental basis for further studying the effect of OR on anti-aging.Objective:①To observe the changes of weight and oestrous cycle in aged rats after taking OR, and analysis the changes of pathology in liver,uterus,ovary,and detecte activity level of SOD,CuZnSOD,POD andT-AOC in aged rats serum and liver.②To detect the level of mRNA cyclinDl and p21 by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and evaluated the expression of p16 and p21 by immunohistochemistry(IHC) in liver,uterus,ovary of aged rats, and examined the expression of cyclinDl,cyclinD3,CDK6 and pRb protein of liver and uterus by Westernblotting, and to further find out the mechanism of OR in anti-aging and the mechanism of OR in delaying reproductive senescence.③To demonstrate correlations of antioxidant effect of OR with expression of regulate key factors in cell proliferation.Methods:①Choosing of the experimental animal and establishment of the aging model.Young female SD rats(n=48)were separated into six groups at random:Normal group,D-gal group(350mg/kg/), positive control group,OR group.The rats in positive control group was Vitamin E(VE,30mg/kg) group.The rats in OR group were redivided into three group:OR high dosage group(OR-H,1.8g/kg),OR medium dosage group(OR-M,0.9g/kg),OR low dosage group(OR-L,0.45g/kg).8 rats were in each group. Young male SD rats (n=32) were separated into four groups at random: Normal group,D-gal group(350mg/kg),VE group(30mg/kg),OR group(0.9g/kg).8 rats were in each group. The sub-acutely aging model rats were induced by D-galactose,and the rats in VE and OR group accepted intragastric administration of VE and OR while accepting D-galactose neck back injection for 42 days.②Experimental procedures. To observe the weight change of aged rats during taking OR. The change of vaginal epidermis keratosis was observed by Wright-Giemsas staining in two weeks before rats were killed.Serum,liver,uterus and ovary tissues of each group were obtained after the rats undergoing experiment for 30d with taking OR. We detected activity level of SOD, CuZnSOD, POD and T-AOC in serum and liver by UV spectrophotometry, and observed the pathological changes of liver, uterus,ovary tissues with hematoxylin and eosin staining by microscope. the gene mRNA expression of cyclinDl and p21 were detected by qRT-PCR in liver, uterus, ovary tissues, and evaluated the expression of p16 and p21 by IHC in liver, uterus, ovary tissues, and examined the protein expression of cyclinDl,cyclinD3,CDK6 and pRb by Westernblotting in liver and uterus. To demonstrate correlations of SOD,POD and T-AOC activity level with expression of regulate key factors p16,p21,cyclinD1, CDK6 and pRb proteins in liver.Results:1,Effect of OR on senescent signs,histomorphology and anti-oxidant index in aging rats.1.1 Result of animal experiment:Rat's general signs:Appearance of D-gal group was dispiriteder, less activity, loss of hair, yellowishewithered hair than Normal group. OR could improve aged rats appearance, activity and its hair color and luster. The weight of Normal group has no difference with D-gal group (P=0.152). The weight of OR-H,M group was significantly decreased than D-gal group (P=0.000, P=0.001), but such effect was not shown in aged male rats (P=0.648). OR could also make shorten female aged rats estrus cycle, lengthen oestrus time (P=0.000).1.2 Result of Pathology:Change of macroscopic anatomy, surfaces of liver in Normal group was smoot and glossy. Color of liver was brownish red and substance was soft and crisp. It was no abnormality seen. Surfaces of liver in D-gal group had white necrotic zone and color of liver was partital white. Compare to Normal group, uterus of D-gal group metratrophia and thinning. Ovary was decreased size and its surfaces had fewer punctiform apophysis. Its color was pallor. Shape of liver in OR group was normal and its color was reder than D-gal group. Uterus of OR group was hypertrophy and ovary enlargement than D-gal group. Color of ovary was reder than D-gal group and surfaces of ovary had many white punctiform apophysis.By microscope, we found structure of liver cell in normal group was normal. Appearance of hepatic cord was regular pattern. Hepatic sinusoid and central veins were normal. Liver cell of D-gal group was loss of volume and cell nuclei. Acidophilia of cytoplasm was higher than Normal group. Some liver cell junction of D-gal group was surface loosening. Hepatic sinusoid was obviously extension and hepatic cord became stenosis. Hepatic sinusoid,hepatic cord and structure,size of liver cell and cell muclei in OR-H group were normal. Liver cell and cell nuclei of volume became large and some liver cell had conjugate nuclei. Hepatic sinusoid,hepatic cord had no obviously abnormality. Structure of endometrium, muscularlayer and perimetrium in Normal group were clearness and epithelial cells aligned regularity. There were abundance glandular organ in uterus. Endometrium of D-gal group had epithelial proliferation and became thinning. Excretion of glandular organ lessened or unobviously. There wwas a great quantity of eosinophile granulocyte infiltrating in intercellular substance. All OR groups had analogical atrophic pathplogy change in uterus with D-gal group. But endometrium pathological change got better than D-gal group. Glandular organ hyperplasia and excretion in uterus were obviously in OR-H and OR-M. Its stromal cells became hypertrophy and hyperplasia. Glandular organ and epithelial in endometrium proliferation in OR-L group were lower than OR-H and OR-M groups. Various developmental stage ovarian follicle and multiple corpus luteum was seen by microscope in Normal group. Multilayer granular cell in ovarian follicle was seen. All layer cells in mature follicle of Normal group were well distributed and regularity. Ovarian follicle of D-gal group was impaired development and number of primary follicle increased, and number of mature follicle and corpus luteum decreaed. Different levels ovarian follicle and mature follicle in OR groups were seen by microscope. Corpus luteum volume became large and multilayer granular cells in ovarian follicle were seen in OR groups.1.3 Result of anti-oxidant index:The activity of SOD, CuZn-SOD, POD and T-AOC of serum and liver tissue in aged rats were significantly decreased than Normal group (P=0.000). All OR groups could significantly increase these indexes of serum and liver tissue (P=0.000), OR-H group was the best in all OR groups.2 Expression of gene mRNA cyclinDl was lower expression and gene mRNA p21 was overexpression in liver, uterus, ovary tissues of aged rats, and OR could up-regulate the expression of cyclinDl and retrieve the overexpression of p21 so as to cell proliferation.2.1 Expression of gene mRNA cyclinDl in liver, uterus, ovary tissues of aged rats.2.1.1 The result of qRT-PCR had shown that gene expression of cyclinDl in liver of D-gal female rat group was marked decreased than normal group (P=0.028). All OR groups could notable increased cyclinD1 mRNA's expression than D-gal group(P=0.016,P=0.007,P=0.000). OR-M group was was the best in all OR groups.2.1.2 The result of qRT-PCR had shown that gene expression of cyclinD1 in liver of D-gal male rats group was significantly lower than normal group (P=0.040). cyclinD1 mRNA's expression of OR group had significantly higher than D-gal group(P=0.000).2.1.3 The result of qRT-PCR had shown that gene expression of cyclinD1 in uterus of D-gal group was marked lower than normal group (P=0.000). Expression of cyclinD1 mRNA in all OR groups was significantly higher than D-gal group (P=0.000, P=0.002, P=0.004). The more dose of OR, the more expression of cyclinD1.2.1.4 The result of qRT-PCR had shown that gene expression of cyclinD1 in ovary of D-gal group was prominently lower than normal (P=0.002). Expression of cyclinD1 in all OR groups was significantly higher than D-gal group (P= 0.000).2.2 Expression of gene mRNA p21 in liver, uterus, ovary tissues of aged rats2.2.1 The result of qRT-PCR had shown that gene expression of p21 in liver of D-gal female group was marked increased than normal group (P=0.000). All OR groups could decrease expression of p21 mRNA than D-gal group (P=0.000).2.2.2 The result of qRT-PCR had shown that gene expression of p21 in liver of D-gal male group was marked increased than normal group (P=0.018). OR group could decreaed expression of p21 mRNA than D-gal group (P=0.001).2.2.3 The result of qRT-PCR had shown that gene expression of p21 in uterus of D-gal female group was marked increased than normal. All OR groups were significantly lower than D-gal group(P=0.000).2.2.4 The result of qRT-PCR had shown that gene expression of p21 in ovary of D-gal female group was significantly increased than normal group (P=0.000). All OR groups could markedly decreased expression of p21 mRNA than D-gal group (P=0.000).3 The expression of p16 and p21 were overexpression in liver, uterus, ovary tissues of aged rats by IHC, and the expression of these regulators proteins could be retrieved by using OR so as to cell proliferation.3.1 The result of IHC had shown the positive production of p16 and p21 were mainly multifocal located in endochylema of liver cell and their distribution were diffuse and focus. Positive cell integral of p16 and p21 in liver tissue of D-gal female aged rats were significantly increased than normal group (P=0.000). After taking OR, the integral of positive cell of p16 in OR-M and OR-L groups was significantly decreased than D-gal group(P=0.001,P=0.000). The integral of positive cell of p21 in OR-H,OR-M and OR-L groups was significantly decreased than D-gal group (P=0.000).3.2 The result of IHC had shown that the positive expression of p16 and p21were mainly multifocal located in endochylema of liver cell, and their distribution was diffuse and focus. Positive cell integral of p16 and p21 in liver tissue of D-gal male aged rats was significantly increased than Normal group (P=0.002). After taking OR, the positive cell integral of p16 and p21 in OR group were significantly decreased than D-gal group (P=0.002,P=0.008).3.3 The result of IHC had shown that the positive staining of the P16 and P21 protein in uterus tissue of D-gal rats was mainly located in cytoplasm, which were on the endometrium, the cytoplasm of epithelial cells and interstitial glands and a few in uterine glandular epithelium, and their distribution was diffuse and focus. The expression of P16 positive cells integral in D-gal group was significantly increased than normal group (P=0.009). Positive cell integral of p21 in D-gal group was significantly increased than Normal group (P=0.000). Positive cell integral of P16 and P21 in all OR groups were significantly decreased than D-gal group (P=0.000, P = 0.001; P= 0.002, P= 0.001; P= 0.000, P= 0.000).3.4 The result of IHC had shown that the positive staining of the p16 and p21 protein in ovary tissue of D-gal rats was mostly expressed diffuse staining in the follicle, oocyte, corpus luteumn, with expression in luteal higher than that in the follicle, and the positive expression was also observed in some mesenchymal cells. The positive cell integral of p16 and P21 protein of ovary tissue in D-gal group was significantly higher than normal group (P= 0.019, P=0.000). Positive cell integral of p16 in ovary of OR-M and OR-L groups was prominently lower than D-gal group.(P=0.004,P=0.002). Positive cell integral of p21 in all OR groups was significantly lower than D-gal group(P=0.000, P=0.001, P=0.000).4 The expression of cyclinDl,cyclinD3,CDK6 and pRb were lower in liver, uterus tissues of aging model by Westernblotting, and the expression of these proteins could be up-regulated by using OR so as to cell proliferation.4.1 The result of Westernblotting had shown that expression of cyclinDl protein in liver of D-gal female rats was decreased than normal group and the difference was significant (P=0.000). Expression of cyclinD1 in all OR groups was increased compared with D-gal group and the difference was significant (P=0.000). Expression of cyclinDl in OR-H group was the highest in all OR groups.Expression of cyclinD3 in liver of D-gal group was markedly lower than normal group(P=0.000). Expression of cyclinD3 in all OR groups was increased compared with D-gal group and the difference was significant (P=0.000). Expression of cyclinD3 in OR-H group was the highest in all OR groups.Expression of CDK6 in liver of D-gal group was significantly lower than normal group(P=0.009).Expreesion of CDK6 in OR-H was significantly higher than D-gal group(P=0.014). Expression of CDK6 in OR-M and OR-L groups was lower than D-gal group.(P=0.000). OR-H group was the highest in all OR groups.Expression of pRb in liver tissue in D-gal group was decreased compared with normal group and the difference was significant (P=0.000). pRb expression in all OR groups was higher than D-gal group (P=0.000). pRb expression of OR-H group was the highest in all OR groups.4.2 Expressions of cyclinDl, cyclinD3, CDK6 and pRb in liver tissue of D-gal male group were significantly lower than normal group (P=0.000). Expressions of cyclinD1, cyclinD3, CDK6 and pRb in OR group were significantly higher than D-gal group (P=0.000).4.3 Expression of cyclinDl in uterus tissue of D-gal group had no difference with normal group(P=0.257). Expression of cyclinDl in OR-H, L group were higher than that in D-gal group and the difference was significant (P=0.000, P=0.001), while expression of cyclin D1 in OR-M group had no difference with D-gal group(P=0.999). Expression of cyclinDl in OR-H group was the highest.Expression of cyclinD3 in uterus of D-gal group was significantly lower than normal group (P=0.000). Compared with D-gal group, all OR groups could elevate cyclinD3 expression and the difference was significant (P=0.000). Expression of OR-L was the highest.CDK6 expression in D-gal group was decreased compared with the normal group and the difference was significant (P=0.000). Expression of CDK6 protein in all OR groups were significantly higher than D-gal group (P=0.000). Expression of OR-L was the highest.Expression of pRb in uterus tissue in D-gal group was decreased compared with normal group which was significant (P=0.000). pRb expression in all OR groups was increased compared with D-gal group(P=0.001,P=0.002, P=0.000).5 Correlations were analyzed between antioxidant indexes (activity of SOD, POD and T-AOC) and expression of cell proliferation regulating factors (p16, p21, cyclinDl, CDK6 and pRb protein) in liver tissue.5.1 The negative correlations were demonstrated between the activity of SOD and the expression of p16 protein (Spearman r=-0.487, P=0.000), between the activity of SOD and the expression of p21 protein (Pearson r=-0.849, P=0.000) in liver of female aged rats. The positive correlations were showed between the activity of SOD and the expression of cyclinDl protein (Pearson r=0.752,P=0.000), between the activity of SOD and the expression of CDK6 protein (Spearman r=0.484, P=0.000), also existed activity of SOD and expression of pRb protein in liver tissue of female aged rats (Pearson r=0.740,P=0.000).The negative correlations were demonstrated between the activity of SOD and the expression of p16 protein (Pearson r=-0.719, P=0.000), between the activity of SOD and the expression of p21 protein (Pearson r=-0.680, P=0.000) in liver of male aged rats. The positive correlations were showed between the activity of SOD and the expression of cyclinDl protein (Pearson r=0.755,P=0.000), between the activity of SOD and the expression of CDK6 protein (Spearman r=0.687,P=0.000), also existed activity of SOD and expression of pRb protein in liver tissue of male aged rats (Pearson r=0.863,P=0.000)5.2 The negative correlations were demonstrated between the activity of POD and the expression of p16 protein (Spearman r=-0.403,P=0.000), between the activity of POD and the expression of p21 protein (Pearson r=-0.712, P=0.000) in liver of female aged rats. The positive correlations were showed between the activity of SOD and the expression of cyclinD1 protein (Pearson r=0.760,P=0.000), between the activity of POD and the expression of CDK6 protein (Spearman r=0.396, P= 0.000), also existed activity of POD and expression of pRb protein in liver tissue of female aged rats (Spearman r=0.806,P=0.000).The negative correlations were demonstrated between the activity of POD and the expression of p16 protein (Pearson r=-0.765, P=0.000), between the activity of POD and the expression of p21 protein (Pearson r=-0.774, P=0.000) in liver of male aged rats. The positive correlations were showed between the activity of POD and the expression of cyclinD1 protein (Pearson r=0.869,P=0.000), between the activity of POD and the expression of CDK6 protein (Spearman r=0.716, P=0.000), also existed activity of POD and expression of pRb protein in liver tissue of male aged rats (Pearson r=0.918,P=0.000).5.3 The negative correlations were demonstrated between the activity of T-AOC and the expression of p16 protein (Spearman r=-0.552,P=0.000), between the activity of T-AOC and the expression of p21 protein (Pearson r=-0.764, P=0.000) in liver of female aged rats. The positive correlations were showed between the activity of T-AOC and the expression of cyclinD1 protein (Pearson r=0.577,P=0.000), between the activity of T-AOC and the expression of CDK6 protein (Spearman r=0.357, P= 0.000), also existed activity of T-AOC and expression of pRb protein in liver tissue of female aged rats (Spearman r=0.724,P=0.000).The negative correlations were demonstrated between the activity of T-AOC and the expression of p16 protein (Pearson r=-0.702, P=0.000), between the activity of T-AOC and the expression of p21 protein (Pearson r=-0.703, P=0.000) in liver of male aged rats. The positive correlations were showed between the activity of T-AOC and the expression of cyclinDl protein (Pearson r=0.782,P=0.000), between the activity of T-AOC and the expression of CDK6 protein (Spearman r=0.615, P=0.000), also existed activity of T-AOC and expression of pRb protein in liver tissue of male aged rats (Pearson r=0.880,P=0.000).Conclusion:1. Cell morphologic and pathological structure liver, uterus, ovary tissue in aged rats is damaged. Estrouscycle of female rat is obviously prolonged and oestrus becomes shorten. Activity of SOD, CuZnSOD, POD and T-AOC in serum and liver are obviously lower in aged rats. After taking OR, impairment of pathology is able to be restored and OR can maintain cellular normal form, alleviates the pathological change of uterus and ovary. OR can shorten Estrous cycle and prolong oestrus in female aged rats. OR has definite antioxidative effects and enhances antioxidative indexes of serum and liver. These results indicate that OR delays senescence of organism and reproductive organs in aged rats.2. Result of qRT-PCR shows expression of gene cyclinDl mRNA in liver, uterus and ovary tissues of aged rats was lower. Expression of gene p21 mRNA in liver, uterus and ovary tissues of aged rats was overexpression. OR can increase expression of cyclinDl mRNA in liver, uterus and ovary tissues of aged rats. Meanwhile OR also can decrease over-expression of p21mRNA in liver, uterus and ovary of aged rats.3. Result of IHC shows expression of p16 and p21protein in liver, uterus and ovary tissues of aged rats was overexpression. OR can decrease over-expression of p16 and p21 protein in liver, uterus and ovary tissues of aged rats.4. Result of Westerblotting shows expression of cyclinD1, cyclinD3, CDK6 and pRb protein was lower in aged rats. OR can enhance expression of cyclinD1, cyclinD3, CDK6 and pRb in liver, uterus and ovary tissues of aged rats.5. Result of correlation analysis between antioxidation of OR and regulating factors of cell proliferation indicates there is a correlation in both sides, but it has difference in gender. In male aged rats, the negative correlations are demonstrated between the expression of p16 and SOD,POD and T-AOC, between the expression of p21 and SOD,POD and T-AOC. The positive correlations are shows between the expression of cyclinD1 and SOD,POD and T-AOC, between the expression of CDK6 and SOD,POD and T-AOC, also exists expression of pRb and SOD,POD and T-AOC.In female aged rats, the negative correlations are demonstrated between the expression of p16 and SOD,POD and T-AOC, between the expression of p21 and SOD,POD and T-AOC. According to coefficient correlation comparison, p21 correlation was better than p16's. This is one of difference with male rats. The positive correlations are shows between the expression of cyclinDl and SOD,POD and T-AOC, between the expression of CDK6 and SOD,POD and T-AOC, also existed expression of pRb and SOD,POD and T-AOC. CDK6 correlation also is one of difference with male rats.6. We postulate one of the anti-aging mechanism of OR is as below:↓ROS accumulation,↑antioxidizing ability of organism→meanwhile↓p16 or p21 over-expression in aged tissues→↑positivie regulating factors of cell proliferation-cyclinDl,cyclinD3 and CDK6 protein→↑phosphorylation level of Rb protein→dissociation of E2F1 protein→bind to target gene promoter in intranuclear→cell differentiation and proliferation→anti-aging effect, which needs more evidences from experiments.