Effect Of Panax Notoginseng Saponins On Nogo-NgR And RhoA-ROCK2 Signaling Pathway In OGD/R SH-SY5Y Cells

Objective:In this study,we try to find the effect of Panax notoginseng saponins (PNS) on the mRNA and protein expressions of NgR1,RhoA and ROCK2.To explore the mechanism of PNS on Nogo-NgR and downstream Rho-ROCK signaling pathway on OGD/R SH-SY5Y cclls,thereby providing new basic theory on traditional Chinese and Western medicine treatment of cerebral ischemia.Methods:The optimal concentration of Y27632 on protecting SH-SY5Y cells from injury by the CCK-8 method.Oxygen-glucose deprivation and reperfusion (OGD/R) method were adopted to establish model of injured SH-SY5Y cells. SH-SY5Y cells were divided into eight groups, the control group, OGD/R model group, retinoic acid(RA) group, high dose Panax notoginseng saponins (PNS) group(640mg/L), low dose PNS group(320mg/L), Y27632 group, Y27632+ high dose PNS group,and Y27632+ low dose PNS group.MTT assay was used to observe the function of PNS on SH-SY5Y cells. RT-PCR and Western blot analysis were used to detecting the effect of PNS on the mRNA and protein expressions of NgRl,Rho A and ROCK2.Results:1 The optimal concentration of Y27632 was 10μg/mL,and the time point to pretreat stimulated SH-SY5Y cells with oxygen and glucose deprivation was 24h which was superior to 6h and 12h.2 The MTT results shows that the activity of SH-SY5Y cells was significantly decreased in the OGD/R model (P<0.01)compared to that of in the control group.The activity of SH-SY5Y cells in RA group, high dose PNS group, Y27632 group, Y27632+ low dose PNS group and Y27632+ high dose PNS group significantly increased when compared to that of in the OGD/R model group(P<0.01).The activity of cells in the low dose PNS group was also higher than that in the OGD/R model group but there was no statistical significance.The activity of cells in the high dose PNS group was higher compared to the low dose PNS group and Y27632+high dose PNS group was higher than the high dose PNS group(P<0.05).3 (1)The mRNA expressions of NgR1,RhoA and ROCK2 in the OGD/R model increased obviously compared to the control group(P<0.01).(2)RT-PCR shows the mRNA expression of NgRl in each treatment group significantly decreased when compared to the OGD/R model group(P<0.01), but there was no significant difference between the OGD/R model group and the Y27632 group(P>0.05).(3)The expression of RhoA mRNA in the low dose PNS group and the Y27632+ low dose PNS group had significant difference compared to the OGD/R model group(P<0.05). The mRNA expression of RhoA in the RA group,the high dose PNS group and Y27632+ high dose PNS group decreased significantly compared to the model group (P<0.01).(4)The mRNA expression of ROCK2 in each treatment group decreased obviously compared to the OGD/R model group especially in the Y27632 group, Y27632+high dose PNS group, Y27632+low dose PNS group(P＜0.01).4(1)The protein expressions of NgR1,RhoA and ROCK2 in the OGD/R model increased obviously compared to the control group(P<0.01,P<0.05).(2) Western blot shows, the expression of NgRl in each treatment group decreased compared to the OGD/R model group,but only in the Y27632+ low dose PNS group and Y27632+ high dose PNS group showed a significant difference(P<0.01 or P<0.05) compared to the OGD/R model group.(3) The expression of RhoA in each treatment group was decreased significantly compared to the model group(P<0.01),especially in the high dose PNS group, decreased significantly compared to the low dose PNS group and showed a dose dependent trend.(4) The protein expression of ROCK2 in the Y27632 group, Y27632+ low dose PNS group and Y27632+ high dose PNS group decreased significantly compared to the model group (P<0.01 or P<0.05).The protein expression of ROCK2 in the Y27632+ low dose PNS group and Y27632+ high dose PNS group decreased obviously compared to low dose PNS group and Y27632+ high dose PNS group showed significant difference when compared with the high dose PNS group(P<0.05).Conclusion:1 Significantly increased SH-SY5Y cells activity can be acquired at the concentration of 10μg/mL Y27632 and at the time point of 24h before oxygen glucose deprivation stimulation.2 PNS can inhibit the mRNA and protein expression of NgRl and perhaps adjustment the function of RhoA and ROCK2.3 PNS can inhibit OGD/R injured SH-SY5Y cells, thus promote the growth and regeneration of axon.