| Background:Retinitis pigmentosa(RP)is the most representative disease of retinal degeneration,which is characterized by structural and functional abnormalities of pigment epithelium,causing apoptosis and necrosis of photoreceptor cells.The main clinical features are nocturnal blindness,progressive visual field defect,retinitis pigmentation,and waxy atrophy of optic disc.As an alkylated carcinogen,N-methyl-N-nitrosourea(MNU)can selectively damage retinal photoreceptor cells,lead to cell death and induce RP,which has been widely used in RP research.In RP patients,photoreceptor apoptosis and retinal inflammatory factors are up-regulated,so anti-inflammatory and anti apoptosis are the main targets of RP treatment.At present,the main treatment methods of RP are gene therapy,stem cell therapy and drug intervention.However,RP has complex genetic pattern,too many alleles,and there are still technical problems in stem cell transplantation,which make the application of gene therapy and stem cell therapy limited.Therefore,it will be an effective way to search for drugs to treat RP through drug screening,especially traditional Chinese medicine treatment,which is an important possible way and widely valued.Cassiae Semen is famous for its eyesight function.It is an important medicine in the treatment of ophthalmic diseases in traditional Chinese medicine.Cassiae Semen polysaccharide is a kind of water-soluble polysaccharide extracted from Cassiae Semen,which is one of its main active components and also the main component to play the role of eyesight.It can be used to treat cataract,retinitis,optic atrophy,glaucoma,conjunctivitis and other eye diseases,but the therapeutic effect and mechanism of RP have not been studied.Phosphatidylinositol 3-kinase(PI3K)is a kind of lipid kinase,which has the dual activity of phosphatidylinositol kinase and serine / threonine protein kinase.Akt,also known as protein kinase B(PKB),is a serine / threonine protein kinase,a typical downstream active molecule of PI3 K,which can promote the proliferation of cone cells.It is found that PI3K/Akt signaling pathway plays an important role in the occurrence and development of various ophthalmic diseases,which can inhibit cell apoptosis,induce cell proliferation and achieve cell protection.Toll like receptors(TLRs)play an important role in the regulation of inflammatory response.The increase of TLR4 level can induce inflammatory cytokines,including tumor necrosis factor-?(TNF-?)and interleukin-1?(IL-1?)and so on.Kappa B(NF-?B)is a kind of proinflammatory transcription factor related to retinal inflammatory response.After TLR4 receptor is activated,NF-?B can be activated by signaling,which plays an anti-inflammatory and immunomodulatory role.Therefore,the Water-soluble Semen cassiae polysaccharide may play an anti apoptotic and anti-inflammatory role in MNU induced RP by regulating PI3K/Akt and TLR4/NF-?B signaling pathway,and then play a therapeutic and protective role in RP.Objectives:1.To explore the anti apoptosis and anti-inflammatory effects of Water-soluble Semen cassiae polysaccharide on RP in rats,and to find out and confirm the protective effect of Water-soluble Semen cassiae polysaccharide on RP.2.To explore the possible mechanism of anti apoptosis and anti inflammation of Water-soluble Semen cassiae polysaccharide in the treatment of RP.It was verified that the Water-soluble Semen cassiae polysaccharide might play a therapeutic and protective role in MNU induced RP by regulating PI3K/Akt and TLR4/NF-?B signaling pathway,providing a theoretical basis for the water-soluble Semen cassiae polysaccharide to become a potential new drug for the treatment of RP.Methods:1.Sprague Dawley(SD)rats were selected and MNU was injected subcutaneously to induce retinitis pigmentosa.The induced RP rats were given water-soluble Semen cassiae polysaccharide 50 mg / kg,100 mg / kg,200 mg / kg by gavage,and MNU was injected for24 hours and 5 days to detect,including retinal thickness,photoreceptor apoptosis,electroretinogram(ERG),etc.to verify the efficacy and anti apoptosis activity of water-soluble Semen cassiae polysaccharide.The expression levels of Bax and Bcl-2were detected by Western blot,caspase 3 and by ELISA.Lipopolysaccharide(LPS)induced m ü ller cells as model cells in vitro.After 72 hours of administration of50mg/ml,100mg/ml,200mg/ml of water-soluble Semen cassiae polysaccharide,apoptosis was detected by flow cytometry,Bax and bcl-2 protein expression were detected by Western blot,caspase 3 protein expression were detected by ELISA.2.The RP model was given water-soluble Semen cassiae polysaccharide 50 mg / kg,100 mg / kg and 200 mg / kg.The expression of TNF-?,IL-1?,IL-6 and IL-18 were detected by ELISA.The expression of TNF-?,IL-1?,IL-6 and IL-18 were detected by ELISA Kit after LPS induced microglia as model cells in vitro and treated with water-soluble Semen cassiae polysaccharide 50 mg / kg,100 mg / kg,200 mg / mg for 2and 4 hours.3.Gene chip was used to screen apoptosis and inflammation related genes,combined with bioinformatics method to predict the possible related signal pathway.Through clustering analysis,functional analysis,dynamic network analysis and other bioinformatics methods,the key genes and signal pathways that play a key role in MNU induced retinitis pigmentosa of rats regulated by water-soluble Semen cassiae polysaccharide were fully mined,and the protein table of the key genes screened was further analyzed.Then,Western blot was used to detect the expression of related proteins,and to clarify the molecular mechanism of water-soluble Semen cassiae polysaccharide regulating MNU induced retinitis pigmentosa in rats.Results:1.The water-soluble Semen cassiae polysaccharide(50mg/kg,100mg/kg and200mg/kg)can inhibit the pathological structure damage of retina caused by MNU injection 24 hours and 5 days later,and the protective effect is dose-dependent.water-soluble Semen cassiae polysaccharide(100mg/kg and 200mg/ /kg)of can significantly increase the retinal thickness.2.The water-soluble Semen cassiae polysaccharide(100mg/kg and 200 mg/kg)can significantly inhibit the apoptosis of photoreceptor cells induced by MNU injection for 24 hours and 5 days.3.In the RP model induced by MNU,the amplitudes of a wave and b wave were decreased,and the amplitudes of a wave and b wave of ERG of Cassiae Semen(50mg/kg,100mg/kg and 200mg/kg)at 24 h and 5 d after MNU injection were increased.4.24 hours and 5 days after the establishment of the model,the water-soluble Semen cassiae polysaccharide participated in reducing the apoptosis of photoreceptor cells of retina by regulating Bax,Bcl-2,Caspase-3 apoptotic protein(Bax,Casepase-3,expression decreased,BCL2 expression increased).In vitro,the water-soluble Semen cassiae polysaccharide was also involved in the reduction of apoptosis rate and cytotoxicity(LDH levels)by regulating Bax,Bcl-2,caspase-3 and apoptosis proteins.5.At 24 hours and 5 days after the establishment of the model,the level of TNF-?,IL-1?,IL-6 and IL-18 in the retina tissue of water-soluble Semen cassiae polysaccharide decreased in a dose-dependent manner.At the same time,the level of TNF-?,IL-1?,IL-6and IL-18 in the retina cells decreased and also showed a dose-dependent manner after the administration of water-soluble Semen cassiae polysaccharide for 2h and 4h in vitro.6.In the rat model,the water-soluble Semen cassiae polysaccharide increased the expression of PI3 K and Akt protein,and inhibited the expression of TLR4 and NF-?B protein.At the cell level,the water-soluble Semen cassiae polysaccharide also increased the expression of PI3 K and Akt protein and inhibited the expression of TLR4/NF-?B protein.Conclusions:1.The water-soluble Semen cassiae polysaccharide has an obvious protective effect on MNU induced RP.2.The water-soluble Semen cassiae polysaccharide may affect the expression of related apoptotic protein by activating PI3K/Akt pathway,so as to play an anti apoptotic role,prevent the occurrence of RP in rats and play a therapeutic role.3.The water-soluble Semen cassiae polysaccharide may reduce the levels of TNF-?,IL-1 ?,IL-6 and IL-18 by inhibiting the TLR4/NF-?B signal pathway,and then reduce the inflammation of retinal tissue and cells,so as to play an anti-inflammatory role.4.The research results of this topic are helpful to further understand the pathogenesis of RP,which provide experimental basis for clinical application of water-soluble Semen cassiae polysaccharide to prevent and treat RP,and make water-soluble Semen cassiae polysaccharide a potential new drug for the treatment of RP. |
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