Study On The Effects Of MTOR Pathway On Apoptosis Of Schwann Cells In Diabetes

Diabetic peripheral neuropathy(DPN)is one of the most common chronic complications of diabetes mellitus leading to slowed peripheral nerve conduction velocity and reduced nerve action potential amplitude,which causes sensory and motor dysfunction,such as numbness,pain,paresthesia,even muscle weakness and muscle atrophy.DPN affects diabetic patients’ life quality.Unger et al.found that the myelinated nerve fibers in the sural nerves of diabetic rats showed decreased transverse area,which was mainly due to the thinning of myelin rather than the change in axon diameter.Schwann cell,which involves in the formation and regeneration of peripheral nerve myelin sheath,is a key component to maintain physiological function of peripheral nerves.Injury of Schwann cell is one of mechanisms to cause DPN.Apoptosis can be detected from Schwann cells of diabetic animals.Additionally,glucose and some metabolites derived from glucose such as methylglyoxal(MG),advanced glycation end products(AGEs)are the main factors to cause Schwann cell apoptosis in diabetes mellitus.The occurrence of apoptosis can be mediated by a variety of pathways,including mitochondria-mediated apoptotic pathway,endoplasmic reticulum(ER)stress-mediated apoptotic pathway and death receptor-mediated apoptotic pathway.By upregulating the ratio of pro-apoptotic molecules(Bax)to anti-apoptotic molecules(Bcl-2,Bcl-XL)located on mitochondrial membrane,mitochondrial membrane permeability is enhanced and cytochrome C is released into the cytoplasm,leading to cleave caspase-3 to cause apoptosis.Endoplasmic reticulum stress starts with the aggregation of large amounts of unfolded protein in endoplasmic reticulum,which activates IRE1,PERK and ATF6,eventually leading to apoptosis by up-regulating the expression of DDIT3 and XBP-1s.Apoptosis mediated by mitochondria and endoplasmic reticulum stress is reported to involve in Schwann cells apoptosis in diabetes mellitus.The mammalian target of rapamycin(mTOR),a serine/threonine protein kinase,is the core protein of mTOR pathway that participates in the regulating a variety of cellular functions,such as cell proliferation,apoptosis,migration and differentiation.There are two forms of mTOR complexes in mammals: mTOR complex 1(mTORC1)and mTOR complex 2(mTORC2).The mTORC1,which is composed of mTOR,RAPTOR,DEPTOR,PRAS40,mLst8 and FKBP38,can affects the processes of protein translation,mRNA generation and ribosome formation.The mTORC2,which is composed of mTOR,RICTOR,DEPTOR,Protor,m Lst8 and mSin1,can phosphorylate protein kinase B(Akt).Multiple studies have confirmed that the regulation of mTOR pathways affects apoptosis in various tumor cells.RAD001,known as an mTOR inhibitor,can significantly increase the cisplatin-mediated apoptosis of A549 cells.In prostate cancer cell lines,combined blocking mTOR and ERK signaling pathways through chemical inhibitors up-regulated the expression of apoptosis factor Bim and inhibited cell proliferation.However,whether mTOR signaling regulates Schwann cells apoptosis process in diabetic peripheral neuropathy and the exact mechanism is not well defined.Therefore,in our study,diabetic mice and high glucose-cultured rat Schwann cells(RSC96)were chosen to detect the expression of apoptosis related proteins(Bcl-2,Bax,cleaved caspase-3,GRP78,DDIT3 and XBP-1)and mTOR pathway factors(mTOR,S6K1 and 4EBP1)in order to determine the effects and mechanism of mTOR pathway on apoptosis of Schwann cells in diabetic peripheral neuropathy.The research paper includes three parts: First,mTOR pathway and the expression of apoptosis related proteins are detected in the sciatic nerves of diabetic mice.Second,the effects of mTOR pathways on apoptosis related proteins are detected in high glucose-stimulated Schwann cells.Third,the effects of mTORC1 and its downstream targets 4EBP-1 and S6K1 on mitochondrial apoptotic pathway are explored in high glucose-treated Schwann cells.Part one The injury of peripheral nerve myelin and the expression of mTOR pathway and apoptosis related proteins in diabetic peripheral neuropathyObjective: To clarify whether there is myelin injury and / or axonal injury in peripheral nerves and the correlation between them,the distal sensory and motor nerve conduction velocity(NCV)and action potential amplitude(AMP)of lower limbs were tested in patients with diabetic peripheral neuropathy.Diabetic mice models were made and followed by the collection of sciatic nerves.The mTOR pathway,mitochondrial apoptosis(Bcl-2,Bax)and endoplasmic reticulum stress mediated apoptosis(GRP78,DDIT3 and XBP-1)were detected to tentatively explore the relationship between mTOR pathway activity and apoptosis in DPN.Methods:1.The electromyogram(EMG),nerve conduction velocity(NCV)and action potential amplitude(AMP)of sural sensory nerve and peroneal motor nerve in the distal lower extremity were tested in 23 diabetic patients with electromyogram/evoked potential detecting instrument.The data correlation was analyzed.2.CD1 mice were intraperitoneally injected with STZ to make type I diabetic models.Being fed for 16 weeks,all mice were deeply anesthetized with chloral hydrate and sacrificed.Sciatic nerves were collected and Western blot was performed to test the expression of phospho-mTOR(Ser2448)and mTOR.TUNEL was used to detect cell apoptosis.The expression level of Bcl-2,Bax,GRP78,DDIT3 and XBP-1 in mouse sciatic nerve was detected by immunofluorescence.The expression level of cleaved caspase-3 was detected by immunohistochemistry.Results:1.The slowed NCV and reduced AMP of sural sensory nerve and peroneal motor nerve in patients with DPN showed significant correlation(P<0.05).2.The activated mTOR decreased by 49.48% in the sciatic nerves of diabetic mice compared with normal mice(P<0.05),whereas the expression of mTOR showed no difference between diabetic mice and normal mice.TUNEL showed the number of apoptosis cells was markedly increased in the sciatic nerves of diabetic mice than normal mice.Decreased Bcl-2 and increased Bax were revealed in the sciatic nerves of diabetic mice compared with normal mice by the method of immunofluorescence.Factors involved in endoplasmic reticulum stress-mediated apoptosis pathway(GRP78 and DDIT3)were significantly increased in the sciatic nerves of diabetic mice.The evident cleaved caspase-3 expression was revealed in the sciatic nerves of diabetic mice by imunohistochemical method.Summary:1.The peripheral nerve myelin injury(showed as slowed NCV)coexisted with the peripheral nerve axon injury(showed as reduced AMP)in Diabetic peripheral neuropathy.The significant correlation between them revealed that the myelin injure and axon injure in DPN may influence each other and developed synchronously.2.The mTOR pathway was significantly inhibited in the sciatic nerves of diabetic mice,manifesting as reduced phospho-mTOR.At the same time,cell apoptosis in sciatic nerves increased and the proteins related to both mitochondrial apoptosis pathway and endoplasmic reticulum stress-mediated apoptosis pathway changed significantly.All the above data indicated that inhibition of mTOR pathway in diabetic mice sciatic nerve might participate in mitochondria and endoplasmic reticulum stress-mediated apoptosis pathway.Part two The effects of mTOR pathway on apoptosis-related proteins in high glucose-stimulated Schwann cellsObjective: The expression of phospho-mTOR and proteins related to mitochondria and endoplasmic reticulum stress-mediated apoptosis were detected in high glucose-stimulated Schwann cells.Furthermore,mTOR pathway was activated or inhibited to detect the changes of apoptosis and related proteins for elucidating eventually the effects of mTOR pathways on apoptosis-related proteins in high glucose-stimulated Schwann cells.Methods:1.Cell culture and groups: RSC96 cells(rat Schwann cells)were cultured in DMEM medium supplemented with 10% serum.(1)In order to explore the effect of high glucose on mTOR pathway and apoptosis,RSC96 cells were divided into three groups: normal glucose group,high glucose group and mannitol group.(2)To study the effect of mTOR pathway inhibition on cell apoptosis,RSC96 cells were randomly divided into four groups: normal group,DMSO group,Torin 1 group(500 nmol/L Torin 1)and rapamycin group(100 nmol/L rapamycin).(3)To elucidate whether the activation of mTOR pathway can reverse high glucose-induced apoptosis,RSC96 cells were divided into three groups: high glucose group,high glucose + DMSO group and high glucose + MHY1485(10 μm MHY1485)group.2.Western blot was performed to test phosphor-mTOR(Ser 2448),mTOR,Bcl-2,Bax,GRP78,DDIT3,XBP-1s and cleaved caspase-3.3.The expression level of cleaved caspase-3 was detected by immunocytochemistry.4.TUNEL was used to detect cell apoptosis.Results:1.High glucose decreased phospho-mTOR and promoted apoptosis in RSC96 cellsWith high glucose treatement for 72 h,RSC96 cells presented a 40.12% decrease in phospho-mTOR(Ser 2448)expression accompanied by a 48.17% decrease in Bcl-2 expression,a 86.53% increase in Bax expression and 1.46 times increase of cleaved caspase-3 expression compared with cells of normal glucose group(P<0.05).ER stress related protein GRP78,DDIT3 and XBP-1s respectively decreased by 45.69%,81.88% and 23.92% in high glucose-stimulated RSC96 cells in comparison with the cells of normal glucose group(P<0.05).The results of immunocytochemistry proved the cleaved caspase-3 expression was significantly upregulated in high glucose-treated RSC96 cells and was mainly located in the nucleus.TUNEL detection further demonstrated that high glucose could cause the elevated cell apoptosis in RSC96 cells.2.The inhibition of mTOR pathway led to mitochondria-controlled apoptosis in RSC96 cellsAs mTOR pathway inhibitors,both Torin 1 and rapamycin decreased significantly phospho-mTOR(Ser 2448)expression compared with normal control cells and DMSO control cells.Bcl-2 was decreased by 40.13% and 43.44% with the treatment of Torin 1 and rapamycin in RSC96 cells versus DMSO-treated group.However,Bax was increased respectively by 78.27% and 71.65%.Immunocytochemistry revealed cleaved caspase-3 expression increased in the nucleus of RSC96 cells with mTOR inhibition.The results of TUNEL revealed that apoptotic cells increased in Torin 1 group and rapamycin group versus normal group and DMSO group.3.Activation of mTOR prevented high glucose-caused apoptosis in RSC96 cellsHigh glucose + DMSO-treated RSC96 cells showed no evident difference in Bcl-2,Bax and cleaved caspase-3 expression in comparison with only high glucose-treated cells(P<0.05).Whereas,mTOR pathway activator MHY1485 increased Bcl-2 expression,decreased Bax and cleaved caspase-3 expression.Immunocytochemistry proved that MHY1485 can inhibit the expression of cleaved caspase-3 in high glucose-treated RSC96 cells.TUNEL also confirmed that MHY1485 decreased cell apoptosis.Summary:1.High glucose caused significant inhibition of mTOR pathways and led to the apoptosis mediated by mitochondrial pathway rather than endoplasmic reticulum stress pathway.2.The mTOR pathways inhibition may lead to mitochondrial pathway related apoptosis in Schwann cells.3.The activator of mTOR pathway could inhibit mitochondrial apoptosis pathway and reverse high glucose-induced apoptosis in Schwann cells.Part three The effects of mTORC1 and its downstream targets on mitochondrial pathway-mediated apoptosis in high glucose-stimulated Schwann cellsObjective: The experiments above proved that high glucose significantly inhibited mTOR pathway in Schwann cells,which led to mitochondrial pathway-mediated apoptosis.In this section,we respectively regulate mTORC1,mTORC2 and their downstream targets to clarify the downstream regulation network of mTOR pathway involved in mitochondrial pathway-mediated apoptosis in high glucose-stimulated Schwann cells.Methods:1.Cell groups:(1)To distinguish the effect of mTORC1 and mTORC2 on cell apoptosis,RSC96 cells were divided into four groups: normal control group,control siRNA transfection group,RAPTOR siRNA transfection group and RICTOR siRNA transfection group.(2)In order to clarify the downstream targets of mTORC1 in high glucose-treated cells,RSC96 cells were divided into three groups: normal glucose group,high glucose group and mannitol group.(3)To illustrate the role of S6K1 in high glucose-induced apoptosis,RSC96 cells were divided into three groups: normal glucose + S6K1 wild type-plasmid transfected group,high glucose + S6K1 wild type plasmid-transfected group and high glucose + S6K1 constitutively activated plasmid-transfected group.2.Western blot was performed to test Bcl-2,Bax,cleaved caspase-3,phospho-S6K1,S6K1,phospho-4EBP1 and 4EBP1.3.TUNEL was used to detect the cell apoptosis.Results:1.The effect of RAPTOR or RICTOR inhibition on Bcl-2,Bax and cleaved caspase-3 in RSC96 cellsWestern blot results showed that RAPTOR si RNA led to a decrease in Bcl-2 expression and an increase in Bax and cleaved caspase-3 expression in RSC96 cells,whereas RICTOR siRNA transfection didn’t cause any changes of Bcl-2,Bax and cleaved caspase-3 expression.2.The effect of high glucose on the phosphorylation of m TORC1 downstream targets S6K1 and 4EBP1 in Schwann cellsWestern blot showed a decrease in phospho-S6K1(Thr 389)expression without any significant change in S6K1 expression.There was no evident difference in 4EBP1 and phospho-4EBP1(Thr 37/46)expression among the cells of normal glucose group,high glucose group and mannitol control group(P>0.05).3.Activation of S6K1 prevented RSC96 cells from high glucose-induced apoposis.In wild type S6K1 plasmid(pRK7-HA-S6K1-WT)-transfected RSC96 cells,high glucose caused a decrease in Bcl-2 and an increase in Bax and cleaved caspase-3.On the contrary,in constitutively activated S6K1 plasmid(pRK7-HA-S6K1-E389-deltaCT)-transfected RSC96 cells,high glucose didn’t cause the above related changes.Summary:1.The mTORC1 rather than mTORC2 involved in high glucose-induced Schwann cell apoptosis through mitochondrial pathway.2.As a downstream target of mTORC1,S6K1 participated in high glucose-induced mitochondrial apoptosis in Schwann cells.The activation of S6K1 can reverse high glucose-caused Schwann cell apoptosis.Conclusions:The diabetic peripheral neuropathy can damage both the myelin and axon of peripheral nerves.The myelin injury and axon injury may influence eah other and develop synchronously.Experimental study shows that high glucose can cause mTOR pathway inhibition in Schwann cell,leading to mitochontria-controlled apoptosis indicated in the upregulation of Bax and downregulation of Bcl-2.The mTORC1 and its downstream target S6K1 are main regulator to mediate high glucose-induced Schwann cell apoptosis.Therefore,activation of mTORC1/S6K1 pathway is an effective method to prevent Schwann cell apoptosis in diabetes mellitus.