Study Of The Protective Effect Of You-Gui-Yin On"Neurovascular Unit"in Vivo And In Vitro And Its Correlation With EPO

BackgroundThe theory of Traditional Chinese Medicine(TCM)believes that "the kidney essence fills the brain",which means the kidneys store the essence which fills the brain.Deficiency of kidney essence can cause emptying of the brain and affect the cognitive function.In TCM,the prescriptions of "supplementing kidney essence" are used to treat cognitive impairment caused by "insufficiency of kidney essence"."Neurovascular unit(NVU)" is the basic unit that reflects the dynamic integrity of brain structure and function,and is also the main body of cognitive function.Our research group hypothesized that "Erythropoietin(EPO)may be an important material basis for kidney essence".Earlier studies found that the classic prescription You-Gui-Yin has brain protection and can upregulate EPO expression.In tis study,we use the adenine-induced chronic renal failure rat model and the in vitro NVU model to observe the ameliorative effect of You-Gui-Yin on chronic renal failure rat cognitive impairment and the overall protective effect on NVU in vivo and in vitro,verifying the correlation between the mechanism of Chinese medicine "supplementing kidney essence and benefiting the brain" and the biological effect of EPO.ObjectivesObserve the protective effect of You-Gui-Yin on the injuru of "neurovascular unit"in vivo and in vitro and its correlation with EPO,and explain the biological mechanism of the effect of "supplementing kidney essence and benefiting the brain".Methods1.Reproduction of adenine-induced chronic renal failure rat model and observation of changes in "cerebral neurovascular unit' and EPO.(1)Model replication and model success evaluation criteria:?Modeling method:male SD rats were given 150 mg·kg-1 adenine by gavage once a day for 2 consecutive weeks.?Criteria for judging the success of model:Measure the content of creatinine in serum and urine,then calculate the endogenous creatinine clearance rate(Ccr),and Ccr<0.21 mL·min-1 is used to judge the successful modeling.?Maintaining of model:Starting from the third week,150 mg·kg-1 adenine were given by gavage once every other day for another 4 weeks.(2)Evaluation of the physique and cognitive function of model rats:urine was collected at the end of 6 weeks of modeling.Morris water maze test and PPI test were performed to evaluate the positioning ability and selective attention ability of rats.After the behavioral tests,animals were anesthetized and blood was taken.The morphology of kidneys were observed.Histomorphology of kidneys' paraffin sections were observed by HE staining.CREA content in serum and urine was measured by automatic blood biochemical analyzer and Ccr was calculated.ACTH,CORT,T content in serum was measured by ELISA.RBC,HGB,HCT in whole blood were detected by blood cell analyzer.(3)Observation of the changes in "neurovascular unit" and EPO of model rats:After the behavioral test,the animals were anesthetized and blood was taken,and the content of EPO in serum was determined by ELISA.The brains of animals were collected and EPO content in brain tissue was detected by Western blotting.Frozen brain slices were made to observe "neurovascular unit" by immunofluorescence.2.Improvement effect of You-Gui-Yin on chronic renal failure rats and its correlation with EPO.(1)Animal grouping and administration:After adenine gavage for 2 weeks,the model rats were randomly divided into model group and You-Gui-Yin 8 g·kg-1,16 g·kg-1,32 g·kg-1 groups.From the third week onwards,rats in each You-Gui-Yin group was gavaged with corresponding dose of You-Gui-Yin everyday for another 4 week.At the same time,model group and You-Gui-Yin groups were given 150 m g·kg-1 adenine every other day to maintain the model for another 4 week.(2)Observation indicators and detection methods:The detection methods of animal physique,cognitive function,"neurovascular unit" and EPO in each group are the same as above.The relative expression of EPOR,P-EPOR,JAK2,P-JAK2,STAT3,P-STAT3 protein in cerebral cortex and hippocampus was detected by Western blotting.3.Promotion and protection effect of You-Gui-Yin on "neurovascular unit" in vitro and its EPO correlation.(1)Model replication and grouping:?Using rat primary cerebral cortical microvascular endothelial cells,astrocytes and neurons to replicate the in vitro"neurovascular unit" model.?The established neurovascular unit model was randomly divided into control group,You-Gui-Yin 50 ?g·mL-1,100 ?g·mL-1,200 ?g·mL-1 groups.The control group is routinely cultured,and You-Gui-Yin groups were given the corresponding concentration of You-Gui-Yin and cultivated for 24 hours.?Oxygen-glucose deprivation(OGD)treatment was performed on the established model of"neurovascular unit" with the hypoxia and glucose-deficient culture medium.After 8 hours,the cell number,axon length and cell transendothelial resistance(TEER)value were significantly lower than the normal group,which was used as the standard to judge the successful establishment of the OGD" neurovascular unit" model.?The established neurovascular unit model was randomly divided into control group,OGD model group,You-Gui-Yin 50 ?g·mL-1,100 ?g·mL-1,200 ?g·mL-1 groups.The OGD model group was treated by OGD,and You-Gui-Yin groups was given corresponding concentrations of You-Gui-Yin.(2)Observation indicators and detection methods:?After 24 hours of culture,the cell morphology of each group were observed,the length of neuron axons were measured,the number of microvascular endothelial cells and astrocytes were counted,and the relative expression of EPO and GAP43 were detect by Western blotting.?Observe the cell morphology of each group.The length of neuron axons were measured.The apoptotic rates of astrocytes,BMECs and neurons were measured by flow cytometry.The relative expression of EPOR,P-EPOR,JAK2,P-JAK2,STAT3,P-STAT3,Bal-2,Bax,Caspase3,Cleaved-Caspase3 protein were detected by Western blotting.Results1.Adenine-induced chronic renal failure model rats have significantly abnormal physique,brain tissue and cognitive function.?Compared with the control group,the serum CREA content of the model group was significantly increased,the urine CREA content was significantly decreased,and the Ccr was significantly decreased(all P<0.01),indicating successful modeling.?Compared with the control group,the weight of rats in model group decreased significantly(P<0.01),the kidneys were obviously swollen,pale in color,the glomeruli contracted,the renal medulla showed more vacuoles and a large amount of brown adenine crystals.?Compared with the control group,the contents of ACTH,T,CORT in the serum of rats in model group increased significantly,and the RBC,HGB,HCT levels in the whole blood decreased significantly(all P<0.01).?Compared with the control group,the PPI under 73dB and 82dB of the model group decreased significantly The escape latency was significantly extended on the 4th and 5th days,the number of crossing platforms and the time ratio as well as the distance ratio crossing platform quadrant were significantly reduced(all P<0.01).?Compared with the control group,the "neurovascular unit" in the hippocampus of the model group was obviously damaged:the number of astrocytes increased,the blood vessel density was significantly reduced,and the neuron axon was lost(all P<0.05).?Compared with the control group,the serum EPO content and the EPO content of the cerebral cortex and hippocampus of the model group were significantly reduced(all P<0.01)2.You-Gui-Yin can significantly improve the physique and cognitive function of adenine-induced chronic renal failure model rats.?Compared with the model group,the weight of rats in You-Gui-Yin 8 g·kg-1,16 g·kg-1,32 g·kg-1 groups was significantly increased(P<0.05),the degree of glomerular atrophy was reduced,and renal medullary vacuoles and adenine crystals were reduced.?Compared with the model group,Ccr of rats in You-Gui-Yin 8 g·kg-1,16 g·kg-1,32 g-kg-1 groups was significantly increased,and the contents of ACTH,CORT,and T in serum were significantly decreased.The RBC,HGB and HCT in whole blood of You-Gui-Yin 16 g·kg-1 and 32 g·kg-1 groups increased significantly(all P<0.05).?Compared with the model group,the PPI of rats in You-Gui-Yin 16 g·kg-1 and 32 g·kg-1groups increased significantly.The escape latency was significantly shortened on the 4th and 5th days,the number of crossing platforms and the time ratio as well as the distance ratio crossing platform quadrant were significantly increased(all P<0.01).3.You-Gui-Yin can significantly improve the"neurovascular unit" injury of adenine-induced chronic renal failure model rats.?Compared with the model group,8 g·kg-1,16 g·kg-1,32 g·kg-1 You-Gui-Yin significantly reduced astrocytes in the hippocampus(P<0.05).?Compared with the model group,8 g·kg-1,16 g·kg-1,32 g·kg-1 You-Gui-Yin significantly increased the vascular density in the hippocampus(P<0.05).?Compared with the model group,8 g·kg-1,16 g·kg-1,32 g·kg-1 You-Gui-Yin significantly reduced the loss of neurons in the hippocampus(P<0.05).4.You-Gui-Yin can significantly reverse the adenosine-induced chronic renal failure model rats EPO content and signaling pathway abnormalities.?Compared with the model group,the content of EPO in serum of You-Gui-Yin 8 g·kg-1,16 g·kg-1,32 g·kg-1 groups increased significantly(P<0.05).?Compared with the model group,the content of EPO in the cerebral cortex and hippocampus of You-Gui-Yin 8 g·kg-1,16 g·kg-1,32 g·kg-1 groups were increased significantly(P<0.05).?Compared with the model group,8 g·kg-1,16 g·kg-1,32 g·kg-1 You-Gui-Yin significantly increased the expression of EPOR,P-JAK2,STAT3 and P-STAT3 in hippocampus,and down-regulated the expression of JAK2(all P<0.05).?Compared with the model group,8 g·kg-1,16 g·kg-1,32 g·kg-1 You-Gui-Yin significantly increased the expression of P-EPOR,P-JAK2,P-STAT3 in cerebral cortex.32 g·kg-1 You-Gui-Yin significantly increased the values of P-EPOR/EPOR,P-JAK2/JAK2 and P-STAT3/STAT3.(all P<0.05).5.You-Gui-Yin has significant growth-promoting effects on three kinds of cells in the "neurovascular unit" in vitro model.The in vitro model of "neurovascular unit" co-cultured with brain microvascular endothelial cells(BMECs),astrocytes and neurons was successfully replicated.Compared with the control group,50 ?g·mL-1,100 ?g·mL-1,200 ?g·mL-1 You-Gui-Yin can significantly increase the number of microvascular endothelial cells and astrocytes in the model,significantly increase the length of neuronal axons,and significantly increase the expression of nerve growth proteins GAP43 and EPO(all P<0.05).6.You-Gui-Yin has a significant protective effect on the in vitro model of oxygen-glucose deprivation of "neurovascular unit".?Successfully constructed in vitro model of OGD "neurovascular unit".Compared with the control group,the OGD group had smaller cell body and axon mutations,and decreased axon network density;brain microvascular endothelial cells and astrocyte cell bodies contracted,and some cells appeared shedding.The number of viable astrocytes,neuron axon length and TEER value were significantly reduced(all P<0.01).?Compared with the control group,the length of neuron axons in the OGD group became shorter.The apoptotic rate of astrocytes,BMECs and neurons increased significantly.The expression of anti-apoptotic protein Bcl-2 was significantly reduced,and the expression of pro-apoptotic protein Bax,Caspase3 and Cleaved-Caspase3 increased significantly(all P<0.05).?Compared with the OGD group,the neuronal axons of You-Gui-Yin 50?g·mL-1,100 ?g·mL-1,200 ?g·mL-1 groups increased significantly,the apoptosis rate of three types of cells decreased significantly,the expression of Bcl-2 increased significantly,and the expression of Bax Caspase3 and Cleaved-Caspase3 Significantly reduced(all P<0.05)7.You-Gui-Yin can significantly reverse the oxygen-glucose deprivation of"neurovascular unit" in vitro model EPO and abnormal signaling pathway.?Compared with the control group,the content of EPO in the OGD group decreased significantly(P<0.01);compared with the OGD group,the EPO expression in You-Gui-Yin 50?g·mL-1,100 ?g·mL-1,200 ?g·mL-1 groups increased significantly(P<0.05).?Compared with the control group,the expression of P-EPOR,JAK2,P-JAK2 in the OGD group was significantly reduced,the expression of EPOR and STAT3 was significantly increased,and the values of P-EPOR/EPOR,P-STAT3/STAT3 were significantly reduced(all P<0.01).Compared with the OGD group,100 ?g·mL-1,200 ?g·mL-1 You-Gui-Yin can significantly up-regulate the expression of P-EPOR,JAK2,P-JAK2,down-regulate the expression of pro-apoptotic proteins EPOR,STAT3,and up-regulate the values of P-EPOR/EPOR,P-STAT3/STAT3(all P<0.05).Conclusions1.The adenine-induced chronic renal failure rats have significant cognitive impairment,accompanied by structural damage to the "neurovascular unit" in the hippocampus and significant decrease in EPO content in the body.2.You-Gui-Yin can significantly improve the physique and cognitive impairment of adenine-induced CRF rats,and has a significant protective effect on the"neurovascular unit" in the hippocampus.Its mechanism is correlated with the increase of EPO content in blood and brain and the regulation of JAK2-STAT3 pathway.3.You-Gui-Yin can significantly promote the growth of three kinds of cells and the expression of EPO in the "neurovascular unit" in vitro model.You-Gui-Yin has a significant protective effect on the "neurovascular unit" in vitro model of oxygen-sugar deprivation and its protective mechanism is related to the increase of EPO content and the regulation of JAK2-STAT3 pathway4.The above results suggest that the mechanism of You-Gui-Yin "supplementing kidney essence and benefiting the brain" is related to the biological effect of EPO.