Establishment Of Bmi1 Gene Knockout Rat Model And The Role Of Metformin In Delaying Its Premature Aging

Bmi1,as a polycomb protein,has a transcriptional repressor and is involved in the regulation of cell cycle and cellular senescence.Previous studies have reported that Bmi1 knockout mice exhibit growth retardation,abnormalities of nerves and hematopoiesis,pre-mature osteoporosis,aging of the kidneys,and mitochondrial dysfunction leading to increased DNA damage and increased cell senescence,showing a pre-mature senescence phenotype.Since the rat model is closer to the human model animal than the mouse model,it has advantages in physiological,toxicological and advanced functional studies.We used the second-generation gene editing tool TELEN to construct the Bmi1 knockout(Bmi1^-/-)rat model,phenotypic differences between WT and Bmi1^-/-rats were compared using imaging,histopathology,and molecular biology methods to determine whether Bmi1 knockout results in rat premature aging.Firstly,we detected the expression of Bmi1 protein in WT and Bmi1^-/-rat tissues by Western blot.The results showed that Bmi1 protein was expressed in WT rats but not in Bmi1^-/-rats,indicating that we successfully constructed Bmi1 knockout rat model.Through weight measurement and survival statistics,Bmi1-deficient rats were found to be smaller in size and weight,and the average survival time was only 72days.X-ray radiography,Micro-CT scan and three-dimensional reconstruction detection revealed that Bmi1 deletion caused a significant reduction in the length of long bones,bone mineral density,bone volume,and the number and thickness of trabecular bone;HE staining,histochemical staining with tartrate-resistant acid phosphatase（TRAP）,and image analysis revealed that Bmi1 deletion resulted in a significant decrease in osteoblast bone formation and a significant increase in osteoclast bone resorption.The use of bone marrow mesenchymal stem cells（BM-MSC）culture,cytochemical staining,flow cytometry,and Western blot methods revealed that Bmi1 deletion decreased proliferation of BM-MSCs and differentiation into osteoblasts,increased ROS levels and significantly down-regulated the expression levels of antioxidant enzymes SOD2 and NQO1protein levels.The use of immunofluorescence staining and senescence-associatedβ-galactosidase（SA-βgal）staining assay revealed that Bmi1 deletion significantly increased the percentage ofγ-H2AX and senescence-associatedβ-galactosidase（SA-βgal）positive BM-MSCs;Bmi1 deletion increased significantly DNA damage in rat lung,liver,and kidney tissues detected by immunohistochemical staining for8-OHd G,53BP1 and p-CHK2.The detection of SA-βgal staining revealed that Bmi1deletion increased significantly senescent cells in rat lung,liver,and kidney.These results suggest that Bmi1 deletion can increase BM-MSCs oxidative stress,DNA damage and senescence,inhibit their proliferation and differentiation into osteoblasts,reduce osteoblast bone formation,and promote osteoclast bone resorption,causing rat premature osteoporosis.Depletion of Bmi1 can also cause growth retardation and premature aging in rats by increasing DNA damage and cell senescence in multiple organ tissues.In view of recent studies showing that the anti-diabetic drug metformin has an anti-aging effect,this study propose that metformin can improve premature aging caused by Bmi1 deficiency.To answer this question,4-week-old Bmi1^-/-rats were given metformin by drinking water at a dose of 1 mg/ml,using normal water-drinking WT and Bmi1^-/-rats as controls,the differences in phenotype between the three groups of rats were compared using imaging,histopathology and molecular biology.The results showed that the administration of metformin to the Bmi1^-/-rats resulted in:1)The size of body and the organs of the thymus,spleen,and kidney,and the body weight significantly increased,the average survival time was extended from 72 days to 97 days;2)Length of tibia and growth plate width,bone mineral density,bone volume,number and thickness of trabecular bone,osteoblast number and type I collagen positive area were increased significantly,osteoclast number was decreased significantly;3)ROS levels in BM-MSCs were decreased significantly,and the expression levels of oxidase Nrf2,SOD2,and NQO1 proteins were significantly upregulated;4)The percentages of 8-OHd G,53BP1,and p-CHK2 positive cells and SA-βgal positive cells were significantly decreased in lung,liver,and kidney tissues;5)The expression levels of p16 and p19 proteins and m RNA were significantly down-regulated in bone,lung,liver/kidney tissues.These results suggest that metformin can improve the anti-oxidation ability of BM-MSCs,inhibit bone cell senescence,promote osteoblast bone formation,and inhibit osteoclast bone resorption,thereby improving Bmi1-deficient rat premature osteoporosis.In addition,metformin can also inhibit the premature aging caused by Bmi1 deletion by reducing DNA damage,inactivating the p16 and p19 signaling pathways,and inhibiting cell senescence in multiple organs.This study successfully constructed a Bmi1 knockout rat premature aging model,which will provide a rat model for studying the mechanism of action of anti-aging drugs.This study also preliminarily expounded the mechanism of action of metformin in delaying premature aging of rats caused by Bmi1 deletion,and increased our understanding of the mechanism of action of metformin in anti-aging.