| Background: Amyotrophic lateral sclerosis(ALS)is a destructive neurodegenerative disorder characterized by progressive muscular dystrophy and paralysis that is caused by the progressive and selective loss of motor neurons in the cerebral cortex,brain stem and spinal cord;most ALS patients die of respiratory failure within three to five years.Unfortunately,ALS has not yet been cured,and its exact pathogenesis remains unclear.Approximately 2% of ALS cases are attributed to a mutation in the Cu/Zn superoxide dismutase(SOD1)gene.At present,it is believed that the mutation of hSOD1 protein is misfolded into a group,being cytotoxic,which is one of the mechanisms leading to familial ALS disease.Some studies have identified potential sex differences in the incidence of ALS.These sex differences prompted us to consider the role of estrogen and aromatase in ALS.Indeed,increasing evidence supports a neuroprotective role for estrogen in a variety of neurodegenerative disease models,including ALS.Aromatase is the key enzyme that catalyzes the synthesis of estrogen by androgen,and most of the ideas suggest that aromatase has a neuroprotective effect for its effects on estrogen.In the central nervous system(CNS),circulating estrogen derived from the ovaries,as well as local estrogen synthesized via aromatase may exhibit neuroprotective effects,which include increasing the survival of motor neurons and increasing neurotrophic factors.Classically,neuroprotective female sex hormones activate two nuclear receptors,estrogen receptor-alpha(ER-α)and estrogen receptor-beta(ER-β).In addition,G protein-coupled receptor 30(GPR30),a novel membrane-bound G-protein-coupled receptor expressed in the CNS,has been found to show a high affinity for estrogen.Therefore,GPR30 may participate in the neuroprotective mechanism of estrogen.The study has shown transgenic mice carrying the human SOD1-G93A(hSOD1-G93A)mutation gene display similar clinical and histopathological features as human ALS patients;consequently,hSOD1-G93 A transgenic mice are commonly used as an animal model to study ALS pathogenesis and treatment.Our laboratory has discussed the effect of ovariectomy on the incidence and behavior of hSOD1-G93 A transgenic mice,which has verified that ovariectomy could lead to the earlier onset,but had no effect on the average life span.In our study,significant weight loss and decreased rotarod test times relative to the CON group were observed 4 weeks earlier and 1 week earlier,respectively,in the OVX group compared with the other hSOD1-G93 A groups.The literature contains few reports describing the mechanisms underlying the effects of ovariectomy in this ALS animal model;therefore we performed Western blotting to measure aromatase and estrogen receptor protein expression and also to assess the expression of the inflammatory cytokines tumor necrosis factor-alpha(TNF-α),secreted by M1 microglia;arginase-1 and transforming growth factor-beta(TGF-β),secreted by M2 microglia;the anti-apoptotic marker B-cell lymphoma-2(Bcl-2);and the pro-apoptotic marker Bcl-2-associated X(Bax)in the lumbar spinal cord to determine the mechanism of estrogen-mediated neuroprotection.At the same time,NSC-34 cell is a hybridized cell line,which can propagate and express motor neuronal characteristics.In our laboratory,the NSC-34 cell line with hSOD1-G93 A stably expression has been established.There are few reports to study the expression of aromatase in NSC-34 cells with hSOD1-G93 A stably expression(hSOD1-G93 A cells).Firstly,we explored these problems.Then,the expression of aromatase in the hSOD1-G93 A cells was interfered by Cyp19a1 overexpression or knockdown,so as to study the function of the enzyme.Therefore,the plasmid of Cyp19a1 cDNA(NM-007810)or Cyp19a1 ShRNA was transfected into h SOD1-G93 A cells through the transient transfection.Then we used RT-PCR and Western blotting to detect the mRNA levels of Cyp19a1 and protein levels of aromatase after transfection,to observe the cell numbers and cell damage,so we could assess the effects of aromatase overexpression or knockdown on hSOD1-G93 A cells.Furthermore,we used Western blotting to investigate the levels of cleaved caspase-9,Bcl-2 or Bax,to detect the level of estrogen receptors,with the purpose of observing the effect of the changes in aromatase expression on the apoptosis and estrogen receptors in hSOD1-G93 A cells.Later,the corresponding estrogen receptor inhibitors or estrogen(E2)were used after transfection,and apoptosis factors were detected again,which would help us to understand the mechanism of aromatase effect on hSOD1-G93 A cells.In our study,we aimed to study the aromatase protective effect and mechanism on ALS when the expression level of aromatase increased or decreased,and to provide theoretical basis for studying the pathological mechanism of ALS and providing the effective treatment strategy in the future.Part one The effects of ovariectomy in an hSOD1-G93 A transgenic mouse model of ALSObjective: The purpose of this study was to observe the effects of ovariectomy on the level of aromatase,estrogen receptor,inflammatory and apoptosis cytokines of the lumbar spinal cord,so as to determine the mechanism of estrogen-mediated neuroprotection in hSOD1-G93 A transgenic mice.Methods: We applied female h SOD1-G93 A transgenic mice,the offspring of a transgenic male and a B6/SJL F1 female,as experimental object.At the age of 30 day,the ends of the tails of mice were cut to identify whether the mice carried hSOD1-G93 A gene.Nine h SOD1-G93 A mice at the same age were randomly divided into three groups: the ovariectomized group(OVX),the sham operation group(Sham)and the positive control group(NO-OVX),of three mice per group.At five weeks of age,the mice in the OVX group were anesthetized using 10% chloral hydrate and were bi-laterally ovariectomized.Mice in the Sham group underwent a similar procedure as the OVX group,except the ovaries were left intact.These and another three non-transgenic mice were used for Western blotting.Mice were assessed using Vercelli’s score of 1-5 as the reference standard: a score of 4,which indicated that hind limb tremors appeared when the tail was suspended,was defined as the time of onset.The lumbar spinal cords of the mice were dissected at disease onset and frozen at-80 °C.We used Western blotting to assess the expression of aromatase,ER-α,ER-β,GPR30,arginase-1,TGF-β1,TNF-α,IKKβ,IKBα,Bcl-2 and Bax.Results:1 Ovariectomy reduced aromatase expression of the lumbar spinal cord in hSOD1-G93 A transgenic mice.2 In the lumbar spinal cord,ovariectomy decreased ER-α expression but not ER-β expression,while both ovariectomy and the sham operation decreased GPR30 expression in hSOD1-G93 A transgenic mice.3 Ovariectomy decreased the expression of the anti-inflammatory factors arginase-1,and both ovariectomy and the sham operation reduced the expression of the anti-inflammatory factor TGF-β and increased the expression of the pro-inflammatory factor TNF-α as well as that of IKKβ.4 Ovariectomy promoted apoptosis by down-regulating the expression of the anti-apoptotic protein Bcl-2,while both ovariectomy and sham operation up-regulating Bax expression.Part two The protective effect of Cyp19a1 overexpression on NSC-34 cells with hSOD1-G93 A stably expressionObjective: The purpose of this study was to explore the protective effect of increased aromatase on ALS and its mechanism through the transient transfection of Cyp19a1 cDNA plasmid in NSC-34 cells with hSOD1-G93 A stably expression(hSOD1-G93 A cells).Methods: When the transfection plasmid was prepared,h SOD1-G93 A cells were obtained after digestion by using 0.25% EDTA-pancreatin.Then the cells were evenly inoculated to six orifice plates.After the cells were completely adhered to the wall,the medium was replaced with serum-free DMEM.When the cells reached the confluence of about 60-70%,hSOD1-G93 A cells were transfected with the empty vector or the vector cloned with the Cyp19a1 cDNA by using Lipofectamine TM 2000 transfection reagent,following the manufacturer’ protocol.In the experiment,ICI 182,780 or G15 was added to the serum-free DMEM 30 minutes before transfection.After transfection for 48 hours,the cell viability in the hSOD1-G93 A group,the group transfected by the empty plasmid(CYP-E)and the group transfected by Cyp19a1 cDNA(CYP-OE)was detected respectively.Similarly,after transfection for 48 hours,LDH activity was assessed respectively.After 48 hours of plasmid transfection,the cells were collected to observe the changes of mitochondria,so as to observe the cell status.The relative levels of Cyp19a1 mRNA were detected by RT-PCR after transfection for 48 hours in the CYP-E and CYP-OE groups.Furthermore,the expression of aromatase,ER-α,ER-β,GPR30 and apoptosis factors,such as cleaved caspase-9,Bax and Bcl-2,were quantified by Western blotting.After the intervention by estrogen receptor inhibitor ICI 182,780 or G15,the expression of apoptosis factors were determinated again.Results:1 After transfection for 48 hours,the relative levels of Cyp19a1 mRNA and aromatase protein were significantly increased in the CYP-OE group.2 Compared with other groups,overexpression of aromatase kept the structure of the mitochondria relatively intact and the vacuoles being not obvious,promoted the proliferation of hSOD1-G93 A cells,and reduced cell membrane damage in the CYP-OE group.3 Compared with other groups,overexpression of aromatase reduced the apoptosis of hSOD1-G93 A cells by reducing the expression of cleaved caspase-9 and Bax,increasing Bcl-2 expression levels in the CYP-OE group.4 Overexpression of aromatase in the CYP-OE group raised the expression of ER-α and GPR30,and ICI 182,780 can reverse the anti-apoptosis effect of aromatase.Part three The effect of Cyp19a1 knockdown by ShRNA on NSC-34 cells with hSOD1-G93 A stably expressionObjective: The purpose of this study was to explore the effect of reduced aromatase on ALS and its mechanism through the transient transfection of Cyp19a1 ShRNA plasmid in NSC-34 cells with hSOD1-G93 A stably expression(hSOD1-G93A).Methods: When the transfection plasmid was prepared,hSOD1-G93 A cells were obtained after digestion by using 0.25% EDTA-pancreatin.Then the cells were evenly inoculated to six orifice plates.After the cells were completely adhered to the wall,the medium was replaced with serum-free DMEM.When the cells reached the confluence of about 60-70%,hSOD1-G93 A cells were transfected with the empty vector or the vector cloned with the Cyp19a1 ShRNA by using Lipofectamine TM 2000 transfection reagent,following the manufacturer’ protocol.After transfection for 48 hours,the cell viability was detected in hSOD1-G93 A group,the group transfected by the empty plasmid(E)and the group transfected by Cyp19a1 ShRNA plasmid(Sh RNA)respectively.Similarly,after transfection for 48 hours,LDH activity was assessed respectively.After 48 hours of plasmid transfection,the cells were collected to observe the changes of mitochondria,so as to observe the cell status.The relative levels of Cyp19a1 mRNA in the E and ShRNA group were detected by RT-PCR after 48 hours of transfection.Furthermore,the expression of aromatase,ER-α,cleaved caspase-9 and Bcl-2 were quantified by Western blotting.Then we added Estradiol(E2)to the cells transfected by Cyp19a1 ShRNA,in order to observing the effect of aromatase reduction on apoptosis whether could be reversed by E2.Results:1 After transfection of Cyp19a1 ShRNA for 48 hours,the relative levels of Cyp19a1 mRNA and aromatase protein were significantly reduced in the ShRNA group.2 Compared with other groups,aromatase reduction aggravated mitochondrial being swollen,made the fracture and disappear of mitochondrial crest more obvious,even cavitation formed,decreased the proliferation of hSOD1-G93 A cells,and increased cell membrane damage.3 Compared with other groups,aromatase reduction increased the expression levels of cleaved caspase-9,reduced Bcl2 expression levels and decreased the expression of ER-α.4 Compared with other groups,E2 could reduce the level of cleaved caspase-9 and increase that of Bcl-2 in the ShRNA+E2 group.Conclusions:1 Ovariectomy reduced aromatase and ER-α expression,promoted inflammation and apoptosis by down-regulating arginase-1 and Bcl-2 in the lumbar spinal cord of hSOD1-G93 A transgenic mice.2 Our study showed that overexpression of aromatase resulted from Cyp19a1 cDNA plasmid transfection increased the proliferation and reduced cell damage of hSOD1-G93 A cells,had a protective effect through anti-apoptotic pathway,and the effect of anti-apoptotic was related to ER-α activated.3 Our study showed that decreased aromatase reduced the proliferation and increased cell damage of hSOD1-G93 A cells,promoted apoptosis,decreased ER-α expression,and the effect of induced apoptotic effects could be reversed by E2. |
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