Effect Of Bushen Jianpi Kaixin Formula On Brain Aging In Subacute Aging Model Rats Based On Inflammation And Bax/Bcl-2/Caspase-3 Apoptosis Pathway

Objective:Brain aging is mainly controlled by aging-related genes in vivo,Nuclear factor kappa B（NF-κB）has transcription activation function and is one of the key mediators of aging.NF-k B can not only activate neuroinflammation,but also induce apoptosis.Bax/Bcl-2/Caspase-3 signaling pathway has the greatest influence on cell apoptosis,which is closely related to them.Bushen Jianpi Kaixin formula is effective in treating senile memory disorder and cognitive dysfunction in clinic,but its mechanism of action has not been fully explained.In this study,the effects of Bushen Jianpi Kaixin formula on NF-κB,p-NF-κB and Bax,Bcl-2,Caspase-3 protein expression in cerebral cortex of subacute aging model rats induced by D-galactose were investigated,so as to explore the possible anti-inflammatory and anti-apoptosis mechanism of Bushen Jianpi Kaixin formula in delaying brain aging.Methods:100 SPF male SD rats,aged 3 months,weighing 220±20g.During the experiment,the animals were free to eat and drink,and all the animals were kept in the animal room with constant temperature（22-25）℃and relative humidity of 40-70%.After one week of adaptive feeding,the Y-type water maze method was used for preliminary screening,and those who were correct for more than 12 times in 15 tests and had normal diet and defecation were qualified animals（excluding brain defects and digestive system problems caused by uncontrollable factors）.According to“Pharmacological Experiment”,the aging model of D-gal rats was made.Specific modeling method:D-gal-0.9%normal saline solution was prepared according to the standard of 30mg/m L mass concentration.SD rats were injected intraperitoneally with 300mg/kg D-gal-0.9%saline solution once a day for 42 days.The diet,activity,weight and mental state of rats were observed during modeling.60 successful rats were selected and divided into 6groups according to the random number table,with 10 rats in each group.They are Bu Shen group（BS group）,Jian Pi group（JP group）,Kai Xin（KX group）,Bushen Jianpi Kaixin group（BSJPKX group）,vitamin E group（VE group）and model group（MOD group）.In addition,10 SD rats were used as the blank group（Ctrl group）,and the Ctrl group was injected with the same amount of saline intraperitoneally once a day for 42 consecutive days.The dosage of each Chinese medicine treatment group was determined according to the ratio of rat to human surface area coefficient:BSJPKX group(9.99 g/kg·d^-1),JP group(4.05 g/kg·d^-1),BS group(3.6 g/kg·d^-1),KX group(2.34 g/kg·d^-1)and VE group(13.5 mg/kg·d^-1).Every day,each Chinese medicine treatment group was converted into decoction of Chinese medicine containing raw drug amount,and the blank and model groups were given the same volume of deionized water every day,with a dose of 1m L/100g for 28days.In order to evaluate,analyze and compare the learning and memory abilities of rats in each group,Morris water maze method（Morris water maze,MWM）was used to conduct behavioral tests.The cerebral cortex of rats was taken,half of the experimental animals in each group were perfused with normal saline,and the brain tissue was internally fixed with paraformaldehyde.The average optical density changes of NF-κB,p-NF-κB,Bax,Bcl-2 and Caspase-3 in the cerebral cortex of rats in 7 groups were detected by immunohistochemistry.Results:1.Changes of learning and memory ability of experimental rats in each group.Compared with Ctrl group,the spatial learning and memory ability of rats in MOD group decreased significantly（P<0.01）.Compared with MOD group,the spatial learning and memory abilities of rats in other treatment groups were improved（P<0.05 or P<0.01）.2.Expression changes of morphological changes of cerebral cortex of experimental rats in each group.The results of H＆E staining of rat cerebral cortex in each group showed that neurons in Ctrl group were evenly stained,numerous,well-shaped,orderly and compact,with round nucleus and clear cell membrane and nuclear membrane.In MOD group,neurons in hippocampus were unevenly stained,with few numbers,disordered arrangement and sparse distribution,among which neuron necrosis,nuclear pyknosis,unclear cell membrane and nuclear membrane,and scattered layers were found.Compared with MOD group,the number of neurons in BSJPKX group increased obviously,and its distribution was more regular,and the pathological manifestations of neurons were significantly improved compared with MOD group.Compared with MOD group,the number of surviving neurons in VE group increased,and the arrangement of neurons was more orderly,but some cells were swollen.3.The protein expression of inflammatory factors p-NF-κB/NF-κB in cerebral cortex of experimental rats in each group changed obviously.Compared with Ctrl group,the expression of p-NF-κB in cerebral cortex of rats in MOD group increased significantly（P<0.05）,but the expression of NF-κB had no obvious change.Compared with MOD group,the expression of p-NF-κB in cortex of VE group,BS group,JP group,KX group and BSJPKX group was significantly down-regulated（P<0.05 or P<0.01）,but the expression of NF-κB had no significant change.Compared with BSJPKX group,there was no significant difference in p-NF-κB and NF-κB among VE group,BS group,JP group and KX group.4.Compared with Ctrl group,the expression of Bcl-2 protein in MOD group decreased significantly（P<0.01）;Compared with MOD group,Bcl-2protein expression was significantly increased in BS group,JP group,KX group and BSJPKX group（P<0.05 or P<0.01）.Compared with Ctrl group,Bax protein expression in MOD group was significantly up-regulated（P<0.01）.Compared with MOD group,Bax protein expression decreased in BS group,JP group,KX group and BSJPKX group（P<0.05 or P<0.01）.Compared with Ctrl group,the expression of Bax/Bcl-2 protein in MOD group was significantly up-regulated（p<0.01）.Compared with MOD group,Bax/Bcl-2protein expression was significantly down-regulated in BS group,JP group,KX group and BSJPKX group（P<0.01）.Compared with Ctrl group,the expression of Caspase-3 protein in MOD group decreased significantly（P<0.01）.Compared with MOD group,Caspase-3 protein expression in BSJPKX group showed a downward trend（P<0.05）.Conclusion:1.Bushen Jianpi Kaixin formula can effectively improve the abnormal learning and memory ability of aging model rats.2.Bushen Jianpi Kaixin formula can protect neurons and reduce the degree of neuron necrosis in midbrain cortex of aging model rats.3.The mechanism of Bushen Jianpi Kaixin formula on aging model rats may be that it can inhibit the increase of inflammatory factor NF-κB,then inhibit Bax/Bcl-2/Caspase-3 signaling pathway to reduce cell apoptosis and protect neurons from massive damage,so as to prevent or delay the progressive course of aging.