Study On The Protective Mechanism Of Progesterone Mediated By PGRMC1 On Neuronal Damage Induced By A?_25-35 Based On Proteomics

Progesterone is a classical steroid hormone in the body,not only in the reproductive system,but also in the nervous system.The role of progesterone in the central nervous system is not limited to the control of neuroendocrine regulation and reproductive function,but also affects the mature and differentiation of neurons and glial cells,and the physiological processes of stable synapses.Numerous studies have confirmed that progesterone has neuroprotection in many experimental models for different types of nerve damage,such as traumatic brain injury,spinal cord injury,ischemic stroke,hippocampal neuronal excitotoxic damage,and neurodegenerative diseases.Progesterone exerts neuroprotective effects in the central nervous system through a variety of mechanisms,including protecting the blood-brain barrier,reducing cerebral edema,reducing the inflammatory cascade,and reducing cell necrosis and apoptosis.Current studies suggest that multiple receptors and proteins may be involved in the neuroprotection of progesterone,including the classical nuclear receptor?n PR?,membrane progesterone receptor component1?PGRMC1?,membrane progesterone receptor?m PR?,gamma-aminobutyric acid type A receptor?GABA_A?,etc.PGRMC1 is the first potential progesterone membrane receptor obtained.Its molecular weight is only 28KD.It has a single transmembrane structure in which the intracellular portion contains a potential Src2 and Src3 homology domain that transmits extracellular signals to the cell.The current studies of PGRMC1 found that PGRMC1 has the same key domain as cytochrome b5,binds to heme and activates P450 protein,and plays an important role in drug,hormone and lipid metabolism.PGRMC1 also binds to the plasminogen activator inhibitor RNA binding protein-1?SERBP1?and mediates anti-apoptotic effects.It has also been reported that PGRMC1 can act as an adaptor protein to transport m PRa to the cell surface.However,the exact mechanism by which PGRMC1 mediates progesterone neuroprotection has not been elucidated.In recent years,with the advancement of proteomics technology,especially proteomics based on high resolution and high throughput mass spectrometry techniques have been widely used to identify and quantify proteomes of different tissues,cells or organelles.It is very important for exploring protein biological functions,discovering disease biomarkers,and finding drug targets.In this study,RNA interference technology was used to knockdown the PGRMC1 protein of rat cortical neurons.And A?_25-35 were used to establish AD cell model.Then the proteomics under neuroprotective effect of progesterone on neuronal cells before and after PGRMC1 silencing were analyzed.To search target proteins and signaling pathways of progesterone neuroprotection associated with PGRMC1 protein through bioinformatics.In order to provide an experimental basis for comprehensive and accurate understanding of the mechanism of progesterone as a neurosteroid.Part I Effect of PGRMC1 on progesterone attenuates neuronal injuryinduced by A?_25-35Objective:To study the effect of PGRMC1 on progesterone neuroprotection.Methods:The cortical tissue of newborn SD rats was isolated and cultured in vitro to obtain primary rat cortical neurons.PGRMC1 si RNA lentivirus were constructed and infected with neurons.After treated neuronal cells with A?_25-35 to establish AD cell model,progesterone was added for treatment.The effects of progesterone on A?_25-35 neurotoxicity were studied by Hoechst 33258 staining and CCK-8 assay.Results:1 Identification of PGRMC1 si RNA lentivirusAfter sequencing,it was confirmed that the PGRMC1 si RNA lentivirus designed for three different target sequences were completely consistent with the original design.2 Screening results for lentiviral infection conditionsGreen fluorescence intensity was detected under a fluorescence microscope.It was found that after 72 hours of infection of lentivirus,the fluorescence of each group reached the strongest,and then the fluorescence gradually weakened.When MOI=20,the lentivirus infection rate reached81.3%,and the neuronal cell status was good.This infection condition can be used in subsequent experiments.3 Silencing effect of PGRMC1 si RNA lentivirusRNAi?61633?and RNAi?61634?reduced neuronal PGRMC1 expression.Compared with the control group,RNAi?61634?reduced the expression of PGRMC1 by 67.7%,and the silencing effect was the strongest,while the negative control virus did not affect the expression of PGRMC1.Lentiviral RNAi?61634?can be used in subsequent experiments.4 Effect of PGRMC1 on progesterone against neuronal apoptosis inducedIt was confirmed by Hoechst33258 staining experiments that progesterone was able to counteract A?_25-35-induced neuronal apoptosis via PGRMC1.The results showed that a large number of neurons were apoptotic after 48 hours of intervention with 25?M A?_25-35.After progesterone treatment,the neuronal apoptosis rate of the negative control virus group was significantly decreased,and the apoptosis rate of the 1?M progesterone intervention group was 38.0%,which was significantly lower than that of the A?_25-35 treatment group?60.5%,P<0.01?.In the PGRMC1 si RNA group,there was no significant difference in neuronal apoptosis rate between the high-medium-low concentration of progesterone intervention groups and the A?_25-35 treatment group.The neuronal apoptosis rate of the 1?M progesterone treatment group was significantly different before and after PGRMC1silencing.5 Effect of PGRMC1 on progesterone against the decrease of neuronal survival rate induced by A?_25-35It was confirmed by CCK-8 assay that progesterone was able to counteract the decrease in neuron survival rate induced by A?_25-35 via PGRMC1.The results showed that after 48 hours of treatment with 25?M A?_25-35,the survival rate of neurons was significantly decreased.After progesterone intervention,the survival rate of neurons in the negative control virus group was significantly increased.The survival rate of neurons in the1 ?M progesterone intervention group was 69.1%,which was significantlyPGRMC1 si RNA group,only 10?M progesterone increased neuronal survival.The neuron survival rate of the 1?M and 10?M progesterone treatment groups was significantly different before and after PGRMC1 silencing.Conclusions:1 In this part of the experiment,PGRMC1 si RNA lentiviral?RNAi?61634??was successfully constructed,which can knockdown the PGRMC1protein of primary rat cortical neurons.2 Progesterone can significantly reduce the neurotoxicity of A?_25-35,andthe neuroprotective effect of progesterone is significantly attenuated after neuronal PGRMC1 protein silencing,and PGRMC1 protein mediates the neuroprotective effect of progesterone.Part II Effect of PGRMC1 on proteome of injured neurons induced byA?_25-35 under progesterone treatmentObjective:To seek proteins and signaling pathways of progesterone neuroprotective associated with PGRMC1.Methods:TMT combined with mass spectrometry was used to quantitatively analyze the proteome changes of rat primary cortical neuron cells before and after PGRMC1 silencing.And the bioinformatics analysis of differential proteins were used to seek proteins and signaling pathways of progesterone neuroprotective associated with PGRMC1.Results:1 Mass spectrometry experimental conditions and typical peptide spectrum informationDuring the 140 minutes,the mass-nuclear ratio??m/z?was always within�0.02 Da,and the mass??M?was always within�10 ppm with good precision.The parent ion collides with the inert gas in the high energy collision cell,and the peptide bond breaks into many fragment ions.Wherein,the C-terminal fragment ion of the peptide chain is y ion,and the N-terminal fragment ion is b ion.2 Identification of neuronal proteinsSeparated by nanoscale liquid chromatograph,Orbitrap Fusion mass spectrometry data acquisition and Proteome Discoverer 2.1 software search,a total of 6881 proteins and 92751 peptides were identified,of which 65123were specific peptides.3 Principal component analysis of total proteinThe PCA results showed that the first principal component?PC1?accounted for 58.2%of the total variance,and the second principal component?PC2?accounted for 26.7%of the total variance.The two groups were clearly separated in the PC1 axis direction.These results indicate a significant difference between the two groups.4 Bioinformatics analysis of proteinsGO analysis showed that these proteins were mainly enriched in organelles,cell membranes,extracellular regions,and some proteins of synapse were also enriched.These proteins are mainly involved in biological processes such as cellular component organization or biogenesis,metabolic process.And the protein are involved in molecular functions including protein binding,small molecule binding.5 Bioinformatics analysis of different expressed proteinsA total of 1157 differential proteins were screened,of which 143 proteins were up-regulated and 1014 proteins were down-regulated?si PGRMC1/si Scramble?.GO analysis revealed that these differentially expressed proteins were mainly involved in biological processes such as biochemical regulation,response to stimulation,and signaling pathways.These significantly enriched terms are closely related to the signaling pathways of neuronal cells.The differential protein was analyzed by KEGG pathway and enriched into 24 signaling pathways.The top 20 signaling pathways including axon guidance,glutamatergic synapse,GABAergic synapse,long-term potentiation,Ras signaling pathway.6 Network construction of protein interactionThe protein associated with Ras signaling pathway,which was significantly enriched in the KEGG analysis,was constructed into an interaction network based STRING database.Conclusions:According to proteomic results,progesterone significantly changed the proteome of AD cell model before and after PGRMC1 protein silencing.Bioinformatics analysis suggests that the Ras signaling pathway may mediate the neuroprotective effects of progesterone.Part III Effect of PGRMC1 on Ras signaling pathway mediatingprogesterone against neuronal injury induced by A?_25-35Objective:To investigate the mechanism of Ras signaling pathway in PGRMC1-mediated progesterone neuroprotection.Methods:The role of the Ras signaling pathway in the neuroprotection of progesterone was further confirmed by the Ras signaling pathway inhibitor FTI-277.The pull down assay was used to determine the level of GTP-Ras in neuronal cells;Western blot was used to detect the expression levels of related proteins in Ras signaling pathway.Results:1 Effect of FTI-277 on the neuroprotective effect of progesteroneFTI-277 dose-dependently attenuated the neuroprotective effect of progesterone.Compared with the progesterone-treated group,the neuronal apoptosis rate was significantly increased and the survival rate was significantly reduced in the FTI-277 group.After adding 20?M FTI-277,the neuronal apoptosis rate was 41.6%,and the survival rate was 31.2%.The results suggest that 20?M FTI-277 can significantly inhibit the neuroprotective effects of progesterone.And 20?M FTI-277 can be used in subsequent experiments.2 Detection of activated GTP-Ras protein levelsCompared with the control group,the level of active GTP-Ras protein in the A?_25-35 group decreased significantly,which was about 55.9%of the control group;progesterone significantly increased the activity of GTP-Ras protein.After the PGRMC1 protein silenced,the progesterone-activated Ras protein was significantly attenuated and the GTP-Ras protein level was significantly decreased.The progesterone effect of the si Scramble group was not affected.The FTI-277 significantly attenuated the effects of progesterone.3 Detection of PI3K/Akt signaling pathway-related protein levelsCompared with the control group,the expression level of PI3K protein and Akt phosphorylation in A?_25-35 group were significantly decreased.After the treatment of progesterone,expression level of PI3K protein and Akt phosphorylation were significantly increased.After the PGRMC1 protein silenced,the progesterone effect is significantly attenuated.The progesterone effect of the si Scramble group was not affected.The FTI-277 significantly attenuated the effects of progesterone.4 Detection of Bax/Bcl-2 ratioCompared with the control group,the ratio of Bax/Bcl-2 in the A?_25-35group was significantly increased about 7.9 times;after progesterone treatment,the ratio of Bax/Bcl-2 was significantly lower than the A?_25-35 group.After the PGRMC1 protein was silenced,the progesterone effect was significantly attenuated.The progesterone effect of the si Scramble group was not affected.The effects of progesterone in FTI-277 group was significantly attenuated.5 Detection of expression levels of various proteins associated with activation of Ras signaling pathwayCompared with the control group,the total Ras and Grb2 protein levels in the A?_25-35 group were significantly decreased.After the progesterone treatment,total Ras and Grb2 protein levels were significantly increased.After the PGRMC1 protein was silenced,the effect of progesterone on total Ras and Grb2 protein was significantly attenuated.The effect of progesterone in the si Scramble group was not changed.The FTI-277 group also did not affect the role of progesterone.There was no difference in the expression levels of Sos1protein in each treatment group.Conclusions:Progesterone activates the Ras signaling pathway through PGRMC1 to exert its neuroprotective effects.Progesterone may activate the Ras signaling pathway by increasing the total Ras protein and the expression of the connexin Grb2 by PGRMC1.In summary,this assay systematically studied the changes of expression levels of various proteins in neuronal cells induced by progesterone neuroprotection response to PGRMC1 protein silencing by RNA interference and mass spectrometry-based proteomics techniques,looking for target proteins and signaling pathways of progesterone neuroprotection related to PGRMC1.It was found that PGRMC1 mediates the protective effect of progesterone on neuronal damage induced by A?_25-35 through activating Ras signaling pathway.