填髓通络方调控miR-143-3p/NRG-1轴治疗脑小血管病模型大鼠的作用及机制OA

Objective To investigate the effects and mechanism of Tiansui Tongluo Formula in treating cerebral small vessel disease(CSVD)model rats.Methods Using the random number table method,60 SPF-grade male SD rats were divided into sham operation group(n=9)and modeling group(n=51).In the modeling group,a CSVD rat model was established via permanent bilateral common carotid artery occlusion.The modeling group was further divided into model,Tiansui Tongluo Formula low-dose(14.4 g/kg),Tiansui Tongluo Formula high-dose(28.8 g/kg),and donepezil hydrochloride(0.45 mg/kg)groups using the random number table method,with nine rats per group.Rats in each drug group were gavaged with the respective drugs,whereas those in the sham operation and model groups were gavaged an equal volume of saline,once daily for 28 consecutive days.The Morris water maze test was used to evaluate the learning and memory abilities of the rats;hematoxylin-eosin staining was used to observe pathological changes in hippocampal region;and a dual-luciferase reporter assay verified the targeting relationship between microRNA-143-3p(miR-143-3p)and neuregulin-1(NRG-1).Immunofluorescence staining was used to observe the expression of neuronal nuclei antigen(NeuN)in rat brain white matter and NRG-1 in hippocampal tissue;immunohistochemistry staining was used to observe positive NRG-1 expression in hippocampal tissue,and TUNEL staining was used to observe neuronal apoptosis in rat hippocampal tissue.Western blotting was used to detect NRG-1,phosphatidylinositol 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(Akt),phosphorylated Akt(p-Akt),and B cell lymphoma 2-associated death promoter(Bad)protein expressions in hippocampal tissue.Lastly,real-time quantitative PCR was used to detect miR-143-3p and NRG-1 mRNA expressions in the hippocampus.Results Compared with the sham operation group,the model group rats showed prolonged escape latency,reduced platform crossing times,and shorter time spent in the target quadrant.Pathological damage to hippocampal neurons was evident.The average fluorescence intensity of NeuN in brain white matter and NRG-1 in hippocampal tissue decreased.The average optical density value of NRG-1 in hippocampal tissue decreased,the apoptosis rate of nerve cells increased,and NRG-1,PI3K,p-PI3K,Akt,and p-Akt protein expressions were decreased,whereas Bad protein expression was increased.miR-143-3p mRNA expression was increased,whereas NRG-1 mRNA expression was decreased(P＜0.05).Compared with the model group,rats in the Tiansui Tongluo Formula low-and high-dose groups and the donepezil hydrochloride group showed shortened escape latency,and prolonged time spent in the target quadrant.Pathological damage to hippocampal neurons improved.The average fluorescence intensity of NeuN in brain white matter and NRG-1 in hippocampal tissue was increased,the average optical density of NRG-1 in hippocampal tissue was increased,neuronal apoptosis was decreased,NRG-1,PI3K,p-PI3K,Akt,and p-Akt protein expressions were increased,Bad protein expression was decreased,miR-143-3p mRNA expression was decreased,and NRG-1 mRNA expression was increased(P＜0.05).The dual-luciferase reporter gene assay showed a targeted relationship between miR-143-3p and NRG-1.Conclusion Tiansui Tongluo Formula alleviates hippocampal neuron damage,inhibits cell apoptosis,and reduces neuronal loss by regulating the miR-143-3p/NRG-1 axis and its downstream PI3K/Akt signaling pathway,thereby improving cognitive impairment in CSVD rats.