| ObjectiveRetinal degeneration(RD)is a serious,irreversible and blinding eye disease,which seriously affects the visual function and quality of life of patients.At present,there is no effective method to treat Rd.the final outcome of its development is photoreceptor cell oxidation and apoptosis.Therefore,looking for safe,convenient and effective antioxidant therapy is still the key research field of Rd.In this study,the mice model of RD was induced by N-methyl-N-nitrosourea(MNU)in vivo to explore the therapeutic effect and mechanism of salvianolic acids(Sal A)on RD.In vitro,the protective effect of Sal A on MNU injured 661 W cell line of mouse retina photoreceptor cone cellswasinvestigated preliminarily.MethodsMale C57 BL / 6 mice(7-8 weeks)were randomly divided into five groups including MNU group,MNU+Sal-A(0.5)group,MNU+Sal-A(1.0)group,NC group and NC+ Sal-A group.The first three groups were MNU treatment group,and the mice received a single dose of intraperitoneal injection(ip)of 60mg/kg MNU.The latter two groups were control groups,which received a single dose of equal ip injection of normal saline(NS),containing 0.05% acetic acid.Then they were given the following treatment.MNU group: NS 1ml/kg/d was injected into caudal vein from day 1 to7.MNU+ Sal-A(0.5)group: caudal vein injection of Sal A 0.5mg/kg within 1 hour,and0.5mg/kg/d Sal A was injected into the tail vein of rats from day 1 to 7,as the low dose group of Sal A intervention.MNU+ Sal-A(1.0)group: 1.0mg/kg Sal A was injected into caudal vein within 1 hour,and 1.0mg/kg/d Sal A was injected into caudal vein from day 1 to 7,as the high dose group of Sal A intervention.MNU+Sal-A(0.5)group and MNU+Sal-A(1.0)group were given Sal A intervention 8 times respectively.NC group: NC 1ml/kg/d was injected into caudal vein from day 1 to 7.NC+Sal-A group: 1.0mg/kg/d Sal A was injected into caudal vein from day 1 to 7.On the 7th day after MNU treatment,the mice in each group were examined by electroretinogram(ERG),hematoxylin and eosin(HE)staining in retinal pathological sections,and the expression of S-cone and Mcone protein in retina were detected by immunohistochemistry.The protective effect of Sal A on MNU induced RD was analyzed from retinal function and morphology.On the 3rd and 7th days after MNU treatment,the activity of superoxide dismutase(SOD)and the content of malonic dialdehyde(MDA)were measured.Western blot and reverse transcription polymerase chain reaction(RT-PCR)were used to quantitatively detect the protein and mRNA of apoptosis related genes Bax,Bcl-2 and Caspase-3 in mice retina.661W strain of mouse retinal photoreceptor cone cells was cultured in vitro.After 1 μM MNU treatment,50μM Sal A intervention was given.The growth of 661 W cells was observed and recorded under inverted light microscope,and the cell activity of cells in each group was detected by Methylthiazolyldiphenyl-tetrazolium bromide(MTT)method.ResultsOn the 7th day after MNU treatment,ERG examination of mice retina showed that the amplitude of a/b-wave in MNU treatment group was significantly lower than that in NC group(P< 0.01).After Sal A intervention,although the amplitude of a/b-wave was significantly lower than that in NC group(P< 0.01),it was significantly higher than that in MNU group(P< 0.01),and the amplitude of a/b-wave in high-dose MNU+Sal-A(1.0)group was significantly higher than that in low-dose MNU+Sal-A(0.5)group.There was no significant difference between NC+Sal-A group and NC group(P> 0.05).The pathological sections of mice retina stained with HE showed that the ONL in MNU treatment group was significantly thinner on the 7th day,and the thickness was significantly smaller than that in NC group(P< 0.01).The ONL in MNU group was basically eliminated.Although the retinal ONL thickness in MNU+Sal-A(0.5)and MNU+Sal-A(1.0)groups was also significantly lower than that in NC group(P< 0.01),it was significantly higher than that in MNU group(P< 0.01),and the Sal A low-dose group was lower than that in high-dose group(P< 0.01).Typical red signals were detected in the pathological sections of mice retina by immunohistochemical method in NC group and NC+Sal-A group,showing the expression of S-cone and M-cone cones.In MNU group,there was no typical red signal in retinal.Although the red signal in the retinal of MNU+Sal-A(0.5)and MNU+Sal-A(1.0)groups was weaker than that of NC group,but was significantly stronger than that of MNU group.On the 3rd and 7th day after MNU treatment,the activity of retinal SOD in MNU treatment group decreased significantly(P< 0.01).Although the SOD activity in Sal A treatment group was lower than that in normal NC group(P< 0.01),it was significantly higher than that in MNU group(P< 0.01),and that in high-dose MNU+Sal-A(1.0)group was higher than that in lowdose MNU+Sal-A(0.5)group(P< 0.01).There was no significant difference between NC group and NC+Sal-Agroup(P>0.05).The content of MDA increased significantly on the 3rd and 7th day(P< 0.01).The content of MDA in Sal A treatment group was significantly higher than that in NC group(P< 0.01),but significantly lower than that in MNU group(P< 0.01),and that in high-dose MNU+Sal-A(1.0)group was lower than that in low-dose MNU +Sal-A(0.5)group(P< 0.01).There was no significant difference in MDA level between NC + Sal-A group and NC group(P> 0.05).Western blot and RT-PCR showed that on the 3rd day after MNU treatment,the expression levels of Bax and Caspase-3 in the retina of MNU treatment group were significantly higher than that of normal control NC group(P< 0.01).Although the two Sal A treatment groups were significantly higher than that of NC group(P< 0.01),they were significantly lower than that of MNU group(P< 0.01).MNU+Sal-A(0.5)group was higher than MNU+Sal-A(1.0)group(P< 0.01).The expression of Bcl-2 in the treatment group was significantly lower than that in the treatment group(P<0.01).Both Sal A treatment groups were significantly higher than MNU group(P<0.01),and low-dose MNU+Sal-A(0.5)group was significantly lower than high-dose MNU+Sal-A(1.0)group(P<0.01).There was no significant difference in the expression of three apoptosis genes between NC+ Sal-A group and NC group(P> 0.05).On the 7th day after MNU treatment,there was no significant difference in the expression of three apoptosis related genes between MNU group and NC group(P> 0.05).In the two Sal A treatment groups,the expression of Bax and caspase-3 decreased continuously,and the expression of Bcl-2 increased continuously.There was no significant difference in the expression of three apoptosis genes between NC+Sal-Agroup and NC group(P> 0.05).661W cells cultured in vitro in NC group grew well.After MNU treatment,the cells gradually shrunk,curled and fell off.The cell activity of MNUgroup was significantly lower than that in NC group(P<0.01).After MNU treatment,most 661 W cells in MNU+ Sal-A group still adhered to the wall and grew,and a few cells fell off.Although the cell activity was lower than that in NC group(P<0.01),it was still significantly higher than that in MNU group(P<0.01).The growth state of NC+Sal-A cells added with Sal A in cell culture complete medium was similar to that of NC group,and there was no significant difference in cell activity between NC group and NC+Sal-A group(P> 0.05).It can be seen that Sal A can resist the effect of MNU on the activity of 661 W cells.Conclusions1.Both 0.5mg/kg and 1.0mg/kg Sal A injected into the tail vein of mice can protect the retinal electrophysiological function and histopathological morphology of MNU induced mice RD model.The protective effect of high-dose Sal A is stronger.Therefore,Sal A can reduce the degradation effect of MNU on mice photoreceptors and protect MNU induced mice RD model.And it is speculated that its protective effect may be dose-dependent.2.Sal A can regulate the activity of endogenous antioxidant enzymes SOD and the content of lipid peroxidation product MDA in the retina.Through the study of the expression levels of promoting apoptosisgenes Bax and Caspase-3 and anti-apoptotic gene Bcl-2 in mice retina,it was found that the protective effect of Sala on MNU induced mice retinal degeneration model was closely related to the anti-apoptotic mechanism.3.Sal A can improve the damage of MNU to mouse photoreceptor cone cell line 661 W in vitro and increasethe activity of cells. |
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