Study On The Effect And Mechanism Of NMN On Reproduction And Organ Damage In Obese Female Mice And Diabetic Female Mice

The incidence rate of obesity is rising all over the world.Obesity can interfere with multiple processes related to female reproduction,and the low quality of oocytes in obese women is the main cause of poor reproductive outcomes.In addition to causing damage to reproduction,obesity can also affect other organs,leading to inflammation and functional damage of organs including heart,liver,pancreas,kidney,etc.In addition to obesity,maternal diabetes also has adverse effects on embryonic development and pregnancy outcomes by damaging the quality of oocytes.Moreover,the hyperglycemia caused by diabetes can lead to abnormal glucose,abnormal lipid metabolism and oxidative stress,which will lead to the damage of pancreas,liver,kidney,heart and other tissues and organs,and even organ failure in serious cases.Therefore,it is very important to find new methods to reduce oocyte quality damage and organ damage caused by obesity and diabetes.NMN is a key NAD+ precursor,which can reduce obesity induced diabetes and obesity related pathological conditions,and can effectively improve the oocyte quality and fertility of aging mice.However,at present,the impact of NMN on oocyte quality decline,reproductive capacity damage and organ damage caused by obesity and diabetes has not been clearly reported.In this study,the effects of NMN administration on heart,liver,pancreas,kidney,ovary,oocyte and offspring of obese mice induced by high-fat diet were tested.In order to explore the effects of NMN on oocytes and organ damage in diabetic mice,we constructed type 1 diabetic mouse models and examined the effects of NMN on oocytes and organ damage in diabetic mice.The main research contents and results are as follows:1.Establishment of obese mice model and the effect of NMN on metabolism of obese miceFour-week-old female mice were randomly divided into two groups.One group received 10% fat-energy high-fat diet,and the other group received 60% fat-energy high-fat diet to establish obesity mouse model.The weight of high fat diet group(HFD group)mice injected PBS intraperitoneally continued to increase after 10 days of high-fat diet,while the weight of high fat diet group mice injected NMN intraperitoneally(HFD+NMN group)did not continue to increase under the condition of high-fat diet.Compared with the normal diet group(ND group)mice,the adipocytes of HFD group mice were significantly increased,while compared with HFD group mice,the adipocytes of HFD+NMN group mice were significantly decreased.2.NMN reduces ovarian abnormalities and ovarian inflammation in obese miceThe results of weight and ovarian weighing on mice indicate that NMN could recover the decline of ovarian weight and ovarian index caused by obesity.Compared with ND group mice,the m RNA level of pro-inflammatory-related genes in the ovaries of HFD group mice increased and the m RNA level of follicle-development-related genes decreased,while NMN administration decreased the m RNA level of pro-inflammatory-related genes in the ovaries of obese mice and increased the m RNA level of follicle-development-related genes.These results showed that NMN could restore the weight of ovary and abnormal follicular development in obese mice,and reduce the ovarian inflammatory reaction in obese mice.3.NMN improves the oocyte quality of obese mice and restores the birth weight of offspringIn this study,we evaluated the number of oocytes,oxidation level,mitochondrial function,apoptosis,DNA damage,abnormal ratio of chromosome/spindle and lipid droplet distribution in ND group mice,HFD group mice and HFD+NMN group mice.The results showed that: 1)There was no difference in the number of oocytes in the three groups.NMN administration could restore the spindle and chromosome abnormalities of mouse oocytes caused by obesity.2)Compared with ND group,the level of γ-H2 A.X increased significantly in HFD group,while NMN administration could increase the γ-H2 A.X level.There was no statistical difference in the level of pro-apoptotic gene Bax in the oocytes of ND group and HFD+NMN group,while the expression of Bax in the oocytes of HFD+NMN group was lower than that of ND group and HFD group.3)Compared with ND group,the ROS fluorescence intensity of oocytes in HFD group was higher,while NMN administration significantly reduced the ROS fluorescence intensity of oocytes in obese mice.The expression level of antioxidant gene Sod1 in oocytes of HFD group was lower than that of ND group.NMN administration increased the expression level of Sod1 in oocytes of obese mice.4)The red-green signal ratio of oocytes in HFD group after JC-1 staining was weaker than that in ND group,but there was no significant difference between the red-green signal ratio of oocytes in HFD group and HFD+NMN group.Compared with the ND group mouse oocytes,the mitochondrial fluorescence intensity of HFD group mouse oocytes after Mito Tracker Red CMXRos staining decreased,while NMN administration could enhance the mitochondrial fluorescence intensity of obese mouse oocytes.5)The actin fluorescence intensity on the oocyte membrane in HFD group was lower than that in ND group.NMN supplementation could significantly increase the actin fluorescence intensity.6)The fluorescence intensity of lipid droplets in mouse oocytes of HFD group was slightly higher than that of ND group,while the fluorescence intensity of lipid droplets in mouse oocytes of HFD+NMN group was significantly lower than that of HFD group.7)There was no statistical difference in pregnancy rate and litter size between ND group,HFD group,HFD+NMN group mice after they mate with normal male mice.The birth weight of offspring mice in HFD group was significantly lower than that in ND group,and NMN administration could restore the low birth weight of offspring mice caused by obesity.4.NMN restores pancreas,liver,heart and kidney damage in obese miceIn this study,we carried out weighing,HE staining and q PCR experiments on the pancreas,heart,liver and kidney of ND group mice,HFD group mice and HFD+NMN group mice.The results showed that: 1)Compared with normal mice,the area of pancreatic islets in obese mice increased significantly,and there was some pathological damage.NMN administration could recover the pathological damage of pancreas caused by obesity.2)The liver cells of ND group mice were orderly distributed,uniform in size,without degeneration and necrosis.The liver of HFD group mice showed diffuse fatty degeneration and a large amount of fat drop deposition.Compared with obese mice injected PBS only,NMN administration could significantly reduce the density and quantity of fatty degeneration in liver cells of obese mice and reduce fat drop deposition.Compared with the liver of ND mice,the transcription level of pro-apoptotic gene Bax and anti-apoptotic gene Bcl-2 and pro-inflammatory gene Adgre1,Ccl2 and TNF and IL-1β in the liver of HFD mice increased significantly.The transcription level of Bcl-2 was significantly increased,while NMN administration could significantly increase the transcription level of Bcl-2in the liver of obese mice,and reduce Bax,Adgre1,TNF-α and IL-1 β.The transcription level of Ccl2 and Gal-3 has no statistical significance.3)There was no obvious abnormality in the myocardium of ND group mice.The myocardial cells of HFD group mice were disorderly arranged,and a large number of nuclei were squeezed to the edge,while the myocardial cells of obese mice treated with NMN were orderly arranged,and most of the nuclei were intact.Compared with ND group mice,the transcription level of pro-apoptotic gene Bax in HFD group mice was significantly increased,and the transcription level of anti-apoptotic gene Bcl-2 gene was significantly decreased.NMN administration could reduce the transcription level of Bax in the heart of obese mice and increase the transcription level of Bcl-2 gene.The transcriptional level of proinflammatory genes such as CD11 c,IL-1β and i NOS has no statistical difference between three groups of mice.4)There was no obvious abnormality in the kidney of ND group mice.The pathological changes of glomerulus injury and renal tubule dilation occurred in HFD group mice.NMN administration could improve the above pathological damage in obese mice.Compared with the kidneys of ND mice,there is no abnormal expression of Bax gene and IL-1β gene in the kidneys of HFD mice.The transcription level of the anti-apoptotic gene Bcl-2gene is significantly reduced in kidneys of HFD mice,and the transcription levels of pro-inflammatory gene TNF-α,CD11 c and Gal-3 were significantly increased.Compared with HFD mice,NMN administration can significantly increase the transcription level of Bcl-2 gene in kidney and reduce the transcription level of TNF-α,CD11 c and Gal-3.5.NMN improves the quality of oocytes in diabetic mice,but reduces the number of oocytes after superovulation in diabetic miceCompared with the control group,the number of oocytes in the Diabetic+PBS group after superovulation was significantly lower,while the number of oocytes in the Diabetic+NMN group was lower.The results of DCFHDA staining showed that compared with the control group,the ROS fluorescence intensity of the oocytes in the Diabetic+PBS group was higher,while NMN administration reduced the ROS fluorescence intensity of the oocytes.The results of Mito Tracker Red CMXRos staining showed that the mitochondrial fluorescence intensity of the mouse oocytes in the Diabetic+PBS group did not change significantly compared with that in the Control group,while the mitochondrial fluorescence intensity of the mouse oocytes in the Diabetic+NMN group increased.6.NMN improves the pathological damage of kidney and heart in diabetic mice,but has no significant effect on the pathological damage of liver and pancreasCompared with the Control group mice,the pancreas,liver,kidney and heart of the Diabetic+PBS group mice showed serious pathological damage,while compared with the Diabetic+PBS group mice,the pathological damage of heart and kidney from the Diabetic+NMN group mice was reduced,and the pancreas and liver pathological damage was not significantly reduced.