The Effect Of Asiatic Acid On Lipopolysaccharide-Induced Neuronal Damage In Cultured Hippocampus In Vitro And Its Protective Mechanism

ObjectiveBased on the Nrf/HO-1 signaling pathway,explore the effect and mechanism of asiatic acid on primary cultured hippocampal neurons after inflammation and oxidative stress injury in vitro.MethodsPrimary culture of hippocampal neurons: several SD rat suckling rats within0²4 h were sterilised by soaking in ethanol and placed in sterile PBS,brain tissue was stripped using an animal dissecting microscope under sterile environmental conditions and the hippocampus was isolated,the clipped tissue pieces were digested with 0.25% EDTA-free trypsin and the digestion was terminated with several ml of fetal bovine serum the appropriate dose of trypsin and fetal bovine serum was chosen according to the amount of tissue pieces The cells were then inoculated and placed in an incubator for 7 d before being used for experiments.The first part of the experiment was to observe the effect of different concentrations of ascorbic acid treatment on hippocampal neuronal cells.The experiment was divided into four groups: normal control group: equal amounts of DMEM were added;AA(10,20,40 μmol·L^-1)group: hippocampal neurons were cultured for 7 d with the addition of(10,20,40 μmol·L^-1)of AA for24 h.The effect of different concentration gradients of ascorbic acid on the viability of hippocampal neuronal cells was measured by the MTT assay.The second part was to see whether AA could inhibit lipopolysaccharide-induced inflammatory oxidative damage in cultured hippocampal neuronal cells in vitro.The experiment was designed with six groups: normal control group,hippocampal neuronal cells cultured for 7 d were added to 0.1% dimethyl sulfoxide for 24 h;LPS group: hippocampal neuronal cells cultured for 7 d were added to 10 ug/m L of LPS for 24 h;LPS + AA group: hippocampal neuronal cells cultured for 7 d hippocampal neurons were incubated with(10,20,40 μmol·L^-1)of AA for 6 h and then incubated with 10 ug/m L of LPS for 24 h;AA 20 μmol·L^-1group: 7 d hippocampal neurons were incubated with 20 μmol·L^-1 of AA for 48 h.effect for 48 h.The hippocampal neuronal cells treated with the above groupings were assayed for damage and cell viability by lactate dehydrogenase（LDH）leakage rate and MTT;IL^-1β and TNF-α expression in hippocampal neuronal cell cultures were measured by ELISA;NO content in the supernatant was measured by Griess method;MDA and SOD activity levels in the cells were measured by the kit to assess the cellular In the third part,the first four groups were grouped as in the second part,and the last two groups were Nrf2inhibitor-ML385 and ML385+LPS+AA groups respectively.The expression levels of Nrf2,HO-1,NF-KB and Bcl2 were detected in each group of cells.ResultsNeu N and GFAP expression was measured by immunofluorescence staining of cultured hippocampal neurons at 7 d.The results showed that the purity of our primary hippocampal neuronal cells was around 90%.The results of MTT experiments showed that the viability of hippocampal neurons in the LPS10 ug/m L model group was significantly reduced compared to the normal control group（P<0.05）;compared to the LPS group,the addition of AA(10,20,40μmol·L^-1)was able to inhibit the LPS-induced decrease in hippocampal neuronal cell viability for 6 h（P<0.05）,while AA(40 μmol·L^-1)aggravated the LPS-induced hippocampal neuronal cell viability compared with the LPS group.To determine the inhibitory effect of ascorbic acid on lipopolysaccharide-induced inflammatory damage in hippocampal neuronal cells,the inflammatory factors TNF-α and IL^-1β were detected using the Elisa assay,and the results showed that the inflammatory factors TNF-α and IL^-1β were elevated in the LPS group compared with the normal control group（P<0.05）,while the ad-dition of ascorbic acid 6 h before the addition of LPS(10,20 μmol·L^-1)was able to significantly reduce the expression of inflammatory factors.In addition,we measured malondialdehyde（MDA）and superoxide dismutase（SOD）activities in the cells,and the results showed that LPS resulted in significantly lower levels of SOD activity and higher levels of MDA in the cells compared to the normal control group;while the addition of AA showed that AA was able to significantly increase SOD activity and decrease MDA levels as a way to inhibit lipopolysaccharide-induced oxidative stress damage.We also examined the LDH cell leakage rate and cellular NO levels,and the results showed that both the leakage rate an-d NO levels were increased in the model group,while the leakage rate and NO levels were significantly reduced after the drug intervention.In addition,we further observed the expression levels of related proteins in each group,and the immunofluorescence results showed that the addition of cumic acid(10,20μmol·L^-1)could significantly increase the expression of Nrf2 and HO-1 in hippocampal neuronal cells 6 h before the addition of LPS（P<0.01）.The cytomorphological results showed that hippocampal neurons cultured in primary culture for 7 d had full cell bodies with long and thick protrusions and many branches,resembling a lattice-like structure.In contrast to the normal control group,the protrusions of hippocampal neurons were significantly reduced,shortened and thinned by the addition of 10 ug/m L LPS for 24 h.The cell bodies were swollen and the normal network-like structure was barely visible.The hippocampal neuronal cells cultured for 7 d were incubated for 6 h with ascorbic acid(10,20 μmol·L^-1)and then co-incubated with LPS for 24 h.The results showed that ascorbic acid significantly reduced the LPS induced damage to the protrusions and cytosol of hippocampal neuronal cells.Western Blot analysis showed that the NF-KB protein expression level was increased（P<0.01）,while Nrf2,HO-1,and Bcl2 protein expression levels were decreased（P<0.05）in hippocampal neuronal cells by adding LPS for 24 h compared with normal controls.The addition of AA(10,20 μmol·L^-1)significantly downregulated the LPS-induced increase in NF-KB protein expression levels（P<0.05）and increased Nrf2,HO-1,and Bcl2 protein expression levels（P<0.05）6 h before the addition of LPS.Compared with the AA 20 μmol·L^-1+LPS group,the addition of ML385 μmol·L^-1 inhibitor showed a decrease in Nrf2 and HO-1protein expression levels（P<0.01）,indicating that ML385 inhibitor could block the protective effect of AA on hippocampal neuronal cell injury to some extent.Taken together,these results suggest that AA has a significant protective effect on LPS-induced damage to hippocampal neuronal cells,and this effect may be due to its activation of the Nrf2/HO-1 signaling pathway.ConclusionsAsiatic acid ameliorates lipopolysaccharide-induced inflammation and oxidative stress damage in cultured hippocampal neuronal cells in vitro.It was found that cumic acid could activate Nrf2 and translocate it from the cytoplasm to the nucleus,while inducing HO-1 expression in the cells.We therefore hypothesized that the protective effect of Centella asiatica on Lipopolysaccharide-injured hippocampal neuronal cells might be related to the Nrf2/HO-1 pathway.In addition,Asiatic acid may also reduce NF-KB and increase Bcl2 expression,exerting its protective effects.