| BackgroundThe theory of traditional Chinese medicine believes that the deficiency of kidney essence is the core factor of human aging,and the aging of bone marrow is an important manifestation of deficiency of kidney essence.The first choice for the prevention and treatment of aging and osteoporosis is the therapy of invigorating the kidney and nourishing the essence.One of the classic clinical prescriptions is YGY.The previous research of the research group found that YGY has antioxidant and cerebral protective effects on naturally aging rats,YGY has a significant improvement effect on osteoporosis rats with kidney deficiency,and its drug-containing serum can promote normal BMSCs differentiation.However,the changes of bone marrow proteomics in naturally aging rats have not been reported so far,and it is unknown whether YGY has a protective effect on the bone marrow of naturally aging rats.In this paper,funded by the Chongqing Ke-Wei Joint Chinese Medicine Key Project（ZY201801001）,the differences in bone marrow proteomics of natural aging rats and young rats were compared.Natural aging rats and in vitro aging BMSCs models were used to observe Yougui Yin The protective effect on the bone marrow of naturally aging rats,and the mechanism of action was discussed.Objective1.To study the differences of bone marrow proteomics between natural aging rats and young rats.2.To observe the protective effect of YGY on the bone marrow of natural aging rats and its mechanism.Methods1.Comparison of physical and bone marrow differences between natural aging rats and young rats:（1）Model replication:15-month-old SD rats were normally raised to 22-month-old.（2）Model success criteria:Compared with the average value of the control group of 3-month-old young rats,the contents of ROS and MDA in the serum of each model rat were significantly increased,while the contents of SOD,T-AOC and GSH were significantly decreased.（3）Behavioral test:Appearance state of rats,open field test to evaluate autonomous activity,and new object recognition to evaluate cognitive ability.（4）Observation of organ,liver and kidney function:after behavioral test,blood was collected and animals were sacrificed to obtain internal organs to calculate kidney,spleen and thymus indices;HE staining of kidney and spleen paraffin sections were used to observe histomorphology;automatic biochemical analyzer was used to detect kidney in serum.Functional indicators UREA,CREA,liver function indicators AST,ALT.（5）Blood index detection:blood analyzer detects blood routine index RBC,WBC,HGB,HCT,Lym,Mono.（6）Evaluation of bone marrow morphology:HE stained bone marrow paraffin sections to observe histomorphology.（7）Bone marrow differential protein detection:protein extraction,enzymolysis,labeling,mixing and mass spectrometry analysis were performed on bone marrow by isotope labeling quantitative method to screen differential proteins.GO functional annotation,KOG annotation,Pathway enrichment analysis,and PPI protein interaction analysis were performed on the differential proteins,and the screened core differential proteins were verified by WB.2.The protective effect of YGY on the physique and bone marrow of natural aging rats and its mechanism:（1）Grouping and administration:The rats with successful aging model were randomly divided into model group and Youguiyin 8g·kg-1 group,16 g·kg-1 group,and 32 g·kg-1 group,with 10 rats in each group.After the balance test,there was no significant difference between the groups,and the rats were given intragastric administration once a day,and 10 young rats in the control group were given normal saline by intragastric administration for 60 consecutive days.（2）Detection of behavioral,morphological and blood indexes:the same as（3）（4）（5）in method 1.（3）Detection of serum oxidation indexes:TBA method and WST-1method were used to detect serum MDA and SOD contents,microplate method to detect T-AOC and GSH contents,and Elisa to detect serum reactive oxygen species ROS contents.（4）Detection of bone marrow indexes:HE staining of bone marrow paraffin section to observe histological morphology;Elisa to detect the content of reactive oxygen species ROS in bone marrow;WB to detect the relative expression of bone marrow proteins NRF2,HO-1,NOX2,TFR1 and FTH1.3.The protective effect of YGY on the aging model of BMSCs in vitro and its mechanism Research methods:（1）Isolation and identification of BMSCs:BMSCs were isolated from rat femur by whole bone marrow adherence method,and identified by immunofluorescence and flow cytometry.（2）BMSCs senescence model replication and success criteria:D-galactose was used to induce BMSCs senescence,and SA-β-gal+expression was used to determine the success of the model.（3）Observation indexes and detection methods:MTT method was used to detect the proliferation activity of BMSCs;Alizarin red staining was used to detect the osteogenic differentiation of BMSCs;β-galactosidase staining was used to detect the aging degree of BMSCs;DCFH-DA probe was used to detect ROS activity;WB to detect bone marrow The relative expression levels of proteins NRF2,HO-1,NOX2,TFR1and FTH1.Results1.The physique of the natural aging model rats decreased significantly,and the bone marrow morphology changed significantly:（1）The model was successfully replicated:Compared with the average value of the young control group,the serum ROS and MDA contents of each model rat were significantly increased,SOD,T-AOC,GSH The content was significantly reduced（all P<0.05）,and the model success rate was 78.18%.（2）Significantly decreased physical fitness:Compared with the young control group,the rats in the model group had dry and yellow hair and severe hair loss;the number of autonomous activities,cognitive ability,kidney,spleen and thymus indexes were significantly reduced（all P<0.05）;kidney tissue vacuoles were significantly reduced increased,the spleen tissue surrounding lymphatic sheath structure disorder;WBC,Lym,Mono in whole blood significantly increased,RBC,HGB,HCT significantly decreased（all P<0.05）;Serum UREA,CREA,AST,ALT levels significantly increased（all P<0.05）.（3）Significant changes in bone marrow morphology:compared with the young control group,the fat vacuoles in the bone marrow tissue of the rats in the model group increased significantly,and the trabecular bone was sparse and broken.2.There are significant differences in the expression of oxidized proteins in the bone marrow of natural aging rats and young rats:（1）Proteomic analysis results:Compared with the young control group,there are 285 significantly different proteins in the bone marrow of the model group rats,of which 150 are Up-regulated,135 were down-regulated,and the top 10 with significant differences were:Gtf2h3,Neurl2,RT1-Db,Podxl,Srsf4,Trpa1,Tcf15,Kcnk1,Pik3c2a,Ncam1,mainly involved in immunity,cell adhesion,etc.Differential proteins were mainly enriched in signaling pathways such as cell adhesion,ECM-receptor interaction,and ferroptosis.It is speculated that bone marrow aging may be related to lipid peroxidation and cell adhesion.Four core differential proteins NOX2,HO-1,TFR1 and FTH1 were screened out.（2）The results of animal experiments for differentially expressed proteins:Compared with the young control group,the TFR1 and NOX2 proteins in the bone marrow of the model group were significantly increased,and the FTH1 and HO-1 proteins were significantly decreased（all P<0.05）.3.YGY can significantly improve the physique of natural aging rats:（1）Significantly improve the appearance:Compared with the model group,the appearance of the rats in each YGY group was significantly improved.（2）Significantly improved organ atrophy:Compared with the model group,the thymus index of the YGY 8 g·kg-1group and 16 g·kg-1 group was significantly increased,and the spleen index and kidney index of the 16 g·kg-1 group were significantly increased.increased（all P<0.05）;the vacuoles in the kidney tissue of the rats in each dose group were significantly reduced,and the disorder of the lymphatic sheath structure around the arteries in the spleen tissue was reduced;（3）Significantly improved liver and kidney function:YGY 8 g·kg-1 group,The serum ALT levels in the 16 g·kg-1 group and the 32 g·kg-1 group were significantly decreased,the serum UREA and CREA levels in the 16g·kg-1 group were significantly decreased,and the serum AST content in the 32 g·kg-1 group was significantly decreased（all P<0.05）.（4）Significantly improved blood routine indexes:Compared with the model group,the number of Mono in the YGY 8 g·kg-1 group,16 g·kg-1 group,and 32 g·kg-1 group was significantly decreased,and the HGB content was significantly increased.The content of HCT in 8 g·kg-1 group and 16 g·kg-1 group was significantly increased,the number of WBC and Lymph in 8 g·kg-1 group was significantly increased,and the number of RBC in 16 g·kg-1 group was significantly increased（all P<0.05）.（5）Significantly improved antioxidant function:Compared with the model group,the serum SOD content of YGY 8 g·kg-1 group,16 g·kg-1 group and 32 g·kg-1 group was significantly increased;Serum MDA levels in group 16 g·kg-1 and 32 g·kg-1 were significantly decreased,while GSH and T-AOC levels were significantly increased;serum ROS levels in 8 g·kg-1 and 32 g·kg-1 groups were significantly decreased（all P<0.05）.4.The protective effect of YGY on the bone marrow of natural aging rats is related to the regulation of the expression of oxidation-related proteins:（1）Significantly improved bone marrow morphology:Compared with the model group,the fat vacuoles in the bone marrow tissue of each dose group of YGY decreased,and the number of bone trabeculae increased and fractures decreased.（2）Significantly reduced bone marrow oxidation indexes:The content of bone marrow ROS in YGY 8g·kg-1 group,16 g·kg-1 group and 32 g·kg-1 group was significantly decreased（P<0.05）.（2）Significantly regulated the expression of bone marrow oxidation-related proteins:Compared with the model group,the HO-1 protein in the YGY 8 g·kg-1group,16 g·kg-1 group,and 32 g·kg-1 group was significantly increased;The protein of FTH1 in the 8 g·kg-1 group and the 16 g·kg-1 group was significantly increased;the protein of NRF2 in the 16 g·kg-1 group was significantly increased,and the protein of TFR1 was significantly decreased;the protein of NOX2 in the 8 g·kg-1 group was significantly decreased（all P<0.05）.5.The protective effect of YGY on the aging model of BMSCs in vitro is related to the regulation of the expression of oxidation-related proteins:The protective effect of YGY on the aging model of BMSCs in vitro is related to the regulation of oxidation-related proteins:（1）The aging model of BMSCs was successfully replicated.Compared with the control group,the cell viability decreased significantly with the increase of D-galactose concentration,and the number of SA-β-gal+cells of BMSCs in the 30 g/L D-galactose group increased significantly（P<0.05）.（2）YGY significantly protected the aging model of BMSCs.Compared with the control group,the cell activity and osteogenic differentiation ability of the model group were significantly decreased,and the SA-β-gal+expression and ROS level were significantly increased;compared with the model group,the cell activity and osteogenic differentiation ability of each YGY group were significantly increased,the expression of SA-β-gal+and ROS activity decreased significantly（P<0.05）.（3）YGY significantly regulated the expression of oxidation-related proteins in aging BMSCs.Compared with the control group,the TFR1 and NOX2 proteins in the model group were significantly increased,and the NRF2,HO-1 and FTH1 proteins were significantly decreased（all P<0.05）.Compared with the model group,the FTH1protein of YGY 200μg·m L-1 group,400μg·m L-1 group and 800μg·m L-1 group was significantly increased;the NRF2 protein of 400μg·m L-1 group and 800μg·m L-1group significantly increased;NOX protein in 800μg·m L-1 group was significantly decreased,and HO-1 protein was significantly increased;TFR1 protein in 400μg·m L-1 group was significantly decreased（all P<0.05）.Conclusions1.The general state,behavioral ability,liver and kidney function,and antioxidant capacity of the natural aging model rats were significantly reduced,the main organs were atrophied,and the blood routine indexes and bone marrow morphology were significantly abnormal.2.There are 285 proteomes in the bone marrow of naturally aged rats that are different from those of young rats.These differential proteins are mainly involved in signaling pathways such as cell adhesion,ECM-receptor interaction,and ferroptosis.Four lipid peroxidation core proteins involved in ferroptosis were abnormally expressed,among which TRF1 and NOX2 were significantly increased,and HO-1and FTH1 were significantly decreased.3.Animal experiments have shown that the oxidation-related proteins TRF1 and NOX2 in the bone marrow of natural aging rats were significantly increased,and the expressions of HO-1 and FTH1 were significantly decreased,which was consistent with the results of proteomic comparative analysis,suggesting that bone marrow aging of natural aging rats is related to cellular aging.peroxidation mechanism.4.YGY has a significant protective effect on the physique and bone marrow of natural aging rats.It can improve the general state,organ atrophy,abnormal blood routine,decreased liver and kidney function,and decreased antioxidant capacity in natural aging rats;significantly improve bone marrow tissue morphology,reduce osteoporosis,and reduce bone marrow oxidative damage.Its mechanism is related to the regulation of oxidation-related proteins.related to the expression of NRF2,NOX2,HO-1,TFR1,and FTH1.5.YGY has a significant protective effect on D-gal-induced aging model of BMSCs in vitro.It can improve the cell activity,osteogenic differentiation ability,aging marker expression,and oxidative damage degree of BMSCs aging model in vitro,and its mechanism is related to regulating the expression of oxidation-related proteins NRF2,HO-1,NOX2,TFR1,and FTH1. |
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