Comparison Of Extraction Of Morinda Officinalis How Nano Particles And Their Effects On Apoptosis Of BV2 Cells

ObjectiveMorinda officinalis How is a kind of Chinese herbal medicine.It has the effects of nourishing kidney yang,strengthening muscles and bones,and dispelling rheumatism.It is used in the treatment of Alzheimer’s disease,but the mechanism is unknown.Traditional Chinese medicine has problems such as low bioavailability and side effects.Exosomes can deliver a variety of information molecules to target cells or be used as therapeutic drug carriers,while plant exosomes can detect the active ingredients of homologous plants,regulate across species.they have abundant sources,and can be prepared through mass production.However,the current exosomes extraction method lacks a standardized system,and there are few studies on the extraction method and mechanism of traditional Chinese medicine exosomes.The purpose of this research is to compare the effects of exosomelike nanoparticles extracted from Morinda officinalis How by soaking and juice extraction,so as to explore the establishment of a practical and efficient extraction for traditional Chinese medicine exosomes.Then we use network pharmacology to analyze the targets and pathways of Morinda officinalis nanoparticles on BV2 microglia apoptosis,to explore the mechanism of Morinda officinalis nanoparticles.Finally,cell experiments are conducted to verify the effect of Morinda officinalis nanoparticles on BV2 microglia,providing methodology and theory for the possibility of using exosomes as carriers to improve drug efficacy or targeted therapy basis.methodsIn this study,fresh Morinda officinalis How was processed by four different methods: soaking for 2 hours,high-speed squeezing,medium-speed squeezing and low-speed squeezing.The Morinda officinalis juice was collected and extracted by differential ultra-high-speed centrifugation.For Morinda officinalis exosomes,transmission electron microscopy(transmission electron microscopy,TEM)directly observes the size and morphological structure of the isolated Morinda citrifolia exosomes.Nanosight Tracking Analysis(NTA)identifies the particle size distribution and par tic le co nc entra tio n of Mo ri nda off ici nali s e xosom es.The nanoparticles were labeled with Di L,and the fluorescence signal intensity was measured using an in vivo imaging system after tail vein injection in healthy male C57BL/6 mice.We use network pharmacology to analyze the active ingredients of Morinda officinalis nanoparticles and predict the target genes and pathways that regulate apoptosis.Cultivate BV2 microglia,coculture the nanoparticles extracted by soaking and juice extraction method with BV2 microglia,then collect cells after 24 and 48 hours,and use flow cytometry technique to detect cell apoptosis.resultsOur results show that the fresh Morinda officinalis How treated with2-hour soaking,high-speed juice extraction,medium-speed juice extraction,and low-speed juice extraction can be observed under a transmission electron microscope with typical cup-shaped vesicles.The characteristic morphology of exosomes,NTA test results also show that the diameter of Morinda officinalis exosomes extracted by the four methods is within 200 nm,which is consistent with the size of exosomes.The concentration of Morinda officinalis How nanoparticles extracted by the juice extraction is much higher than the exosomes extracted after 2 hours of soaking,and the difference is statistically significant(S1 vs S3 P=0.0014,S1 vs S4P<0.0001,S1 vs S5 P=0.0159).After different speeds of juice extraction,the Morinda officinalis How exosomes concentration obtained by medium speed extraction was the highest,and the difference was statistically significant(S4 vs S3 P<0.0001,S4 vs S5 P<0.0001,),while high speed and low speed was no statistically significant difference in the concentration of nanoparticles extracted after juice extraction(S3 vs S5 P=0.9863).The size of nanoparticles obtained from soaking for 2 hours and high-speed juice extraction is not statistically significant(P>0.05),but there is a difference between the size of nanoparticles obtained from medium and lowspeed juice extraction,which is statistically significant(P<0.0001).The size of Morinda officinalis How nanoparticles obtained after juice extraction at different speeds was significantly different(P<0.001).Animal experiment shows that nanoparticles are mainly distributed in the brains of mice.According to network pharmacology analysis,the content of Morinda officinalis nanoparticles may be beta-sitosterol,which may be affected by the regulation of p53 signaling pathway or apoptosis pathway through CASP3/BCL2/CASP8/CASP9 Apoptosis of microglia BV2.In the apoptosis experiment,it was found that the nanoparticles had no obvious effect on the apoptosis of BV2 cells.ConclusionThere are nanoparticles in fresh Morinda officinalis How.The concentration of Morinda officinalis How nanoparticles extracted by juice extraction is higher than that of soaking,and the concentration of nanoparticles obtained by medium speed extraction is the highest.The size of nanoparticles extracted by soaking is larger than that by juice extraction.The content of Morinda officinalis exosomes may be betasitosterol,which may be affected by the regulation of p53 signaling pathway or apoptosis pathway through CASP3/BCL2/CASP8/CASP9 to the apoptosis of microglia BV2.The nanoparticles had no obvious effect on the apoptosis of BV2 cells.