Screening Of Active Components Of Rosemary Against Nerve Injury And Its Mechanism Of Action

Objectives:Through the establishment of cell oxidative damage model for experimental research,we screened the compounds with significant activity from a series of monomer compounds systematically studied and isolated from the chemical components of rosemary,and further verified them based on the level of molecular mechanism to clarify their neuroprotective mechanism.It will provide a basic basis for the major industrialization of Rosemary medicinal plant resources in Yunnan and provide new candidate molecules for the research of natural drugs against nerve injury.Methods:1.Firstly,the aboveground parts of rosemary were isolated and identified.Then applied different concentrations of H2O2(0-400 μM)The oxidative damage of SHSY5Y cells was induced by H2O2 for 24 hours,and the concentration of H2O2 induced oxidative damage of SH-SY5Y cells was determined to establish the oxidative damage model of SH-SY5Y cells induced by H2O2.After that,the identified compounds were applied to SH-SY5Y cells with normal growth and H2O2 induced injury(EGCG was used as positive control).The cell activity was detected by CCK-8 to verify whether these compounds have cytotoxicity,the ability to promote cell proliferation and the ability to resist oxidative damage,and the monomer compounds with obvious activity were preliminarily selected.2.Taking the active monomer compound "11,12-diacetyl-carnosol(No.20)"screened in the first part as the research object,the experiment was divided into four groups:the control group,the No.20 group,the H2O2-induced group and the No.20+H2O2 group.ROS level was detected by DCFH-DA fluorescence;JC-1 fluorescence was used to detect the level of mitochondrial membrane potential;TUNEL staining and flow cytometry were used to detect the apoptosis rate;NO,MDA and GSH were detected by enzyme labeling;The expression of apoptosis related genes(Bcl-2,Bax,Cyt C,Caspase-3 and Caspase-9)was detected by Q-PCR and Western blot;The expression levels of Nrf2 in nucleus and cytoplasm and HO-1 were detected by Western blot,and the nuclear metastasis of Nrf2 was observed by immunofluorescence to investigate whether the neuroprotective effect of No.20 is related to scavenging intracellular reactive oxygen species,regulating mitochondrial membrane potential and inhibiting apoptosis.3.We further verified it based on the molecular mechanism level.The expression of Nrf2 was silenced by transfection of Nrf2 siRNA.The success of plasmid transfection was verified by Western blot.The expression levels of Nrf2 and HO-1 protein in cells of each group after transfection were detected,and the expression of Nrf2 in nucleus was observed by immunofluorescence.The changes of cell activity and mitochondrial function after Nrf2 silencing were observed by CCK-8 and JC-1 to verify whether Nrf2 mediates the protective effect of No.20 on oxidative damage of SH-SY5Y cells induced by H2O2.Results:1.28 monomers were extracted from the aboveground part of rosemary,and their structures were identified as abietane diterpenoid(including 9 new compounds).The oxidative damage model of SH-SY5Y cells was established(300 μM H2O2 acted on cells for 24 hours).Compound 1-28 was treated on SH-SY5Y cells alone.The results of CCK-8 showed that most compounds did not cause damage to nerve cells at the test concentration.Compounds 5,21,22 and 24 inhibited the growth of SH-SY5Y cells at low concentration(1 μM)and high concentration(25 μM),compounds 1 and 6 only inhibited the growth of cells at high concentration,and compound 23 only inhibited the growth of cells at low concentration.The results showed that compounds 10,11,13-20,27 and 28 showed good neuroprotective effects at the test concentration;In particular,compounds 10,16,18 and 20 had the same neuroprotective effect as the positive drug EGCG,and the cell survival rate reached more than 80%,which was statistically significant.The new compounds 5,6 and 7 showed a certain degree of neuroprotective effect at the test concentration,but 5 and 6 had a certain cytotoxicity.2.After injured by H2O2(300μM)for 24 hours,the SH-SY5Y cell survival rate decreased and the apoptosis rate increased significantly.Bcl-2 expression decreased,Bax,Caspase-3,Caspase-9,PARP and Cyt C in cytoplasm expression levels increased,ROS and NO levels increased,MMP decreased,GSH expression levels decreased,and MDA increased.Pretreatment with compound 20(No.20)could significantly inhibit the decrease of proliferation activity of SH-SY5 Y cells induced by H2O2,reduce the ratio of Bax/Bcl-2,inhibit the expression of Caspase-3,caspase-9 and PARP,inhibit the decrease of MMP and the release of Cyt c,reduce the levels of ROS and NO,increase the expression of GSH and decrease the expression of MDA.In addition,pretreatment No.20 also led to nuclear translocation of Nrf2 and increased the expression of HO-1 protein in SH-SY5Y cells.3.After silencing the expression of Nrf2 by small interfering RNA(siRNA),the expression of Nrf2 and HO-1 in the nuclei of each group were down regulated.After transfection,the survival rate and mitochondrial membrane potential of SH-SY5Y cells injured by H2O2 decreased.Conclusion:1.Most of the 28 rosin diterpenes extracted from rosemary have certain neuroprotective effects and do not damage nerve cells.C-10 carboxyl group or formyl group is the necessary group for the neuroprotective effect of rosin alkyl diterpenes,and the degradation of 10 aldehyde group,20 hydroxyl group or C-20 position is the reason for its cytotoxicity.Acetylated derivatives are more stable and have higher biological activity than precursors,in which No.20 showed better antioxidant damage.2.No.20 can inhibit the loss of activity of SH-SY5Y cells induced by H2O2,prevent its apoptosis,and improve mitochondrial function damage through antioxidant effect.3.No.20 inhibited apoptosis and mitochondrial function damage of SH-SY5Y cells induced by H2O2 through Nrf2/HO-1 pathway.