| Backgroud:Glaucoma is hallmarked with the death of retinal ganglion cells,which results in irreversible vision loss.Many studies have been carried out on glaucoma,but there is no effective and reliable treatment to prevent optic nerve injury.Traditional Chinese medicine has obvious advantages in the treatment of glaucomatous optic nerve injury,but the mechanism is not clear.Our study purposed to explore the underlying molecule mechanism of soothing the liver and unblocking the orifices method in treating glaucoma and protecting optic nerve.Methods1.Primary isolation and purification of RGCs from SD neonatal rat.2.The RGCs apoptosis models were established by L-glutamate in vitro,while morphological changes,activity and apoptosis of RGCs were observed by inverted microscope,CCK-8 detection and annexin V-FITC/PI detection.3.Different concentrations of serum containing traditional Chinese medicine were prepared and CCK-8 detection was used to observe the effect of serum containing traditional Chinese medicine on the activity of RGCs purified and cultured in vitro.4.The purified RGCs were randomly divided into 6 groups,including blank group,model group,low,medium and high dose Chinese medicine serum intervention group and VB12 intervention group.The RGCs in the blank group were cultured for 48 hours with the serum of rats without drugs,while in model group,low,medium and high dose Chinese medicine serum intervention group and VB12 intervention group were treated with 3.2mmol/l L-glutamate for 24 hours firstly and then added respectively with the serum of rats without drugs,low,medium and high dose Chinese medicine serum and VB12 mother liquor for 24 hours,all of which were diluted by 10 times with cell maintenance medium.CCK-8 and annexin V-FITC/PI detection were used to detect the activity and apoptosis rate of RGCs.5.The purified RGCs were randomly divided into 4 groups,including blank group,Chinese medicine serum intervention group and VB12intervention group.The RGCs in the blank group were cultured for 48 hours with the serum of rats without drugs,while in model group,Chinese medicine serum intervention group and VB12 intervention group were treated with 3.2mmol/l L-glutamate for 24 hours firstly and then added respectively with the serum of rats without drugs,high dose Chinese medicine serum and VB12 mother liquor for 24 hours,all of which were diluted by 10 times with cell maintenance medium.The total RNA of RGCs in each group was extracted and hybridized with mi RNA chip to screen the mi RNA differentially expressed.Then,q RT-PCR was used to verify the results.The target genes were predicted by Tar Pmi R and mi RTarget prediction software.Go and KEGG enrichment analysis were used to predict the related pathways.Results1.RGCs purified and cultured in vitro survived for about 7 days,and the cell purity was over 90%.2.Apoptosis of RGCs in vitro could be induced by L-glutamate with concentration above 0.3mmol/l for more than 12 hours.The survival rate of RGCs increased and the apoptosis rate decreased obviously when RGCs were induced by0.5,0.8,1.6,3.2 and 6.4mmol/L L-glutamate.The RGCs survival rate of 55.75±6.11%,induced by 3.2mmol/L L-glutamate for 24h,was suitable for the apoptosis model in subsequent experiments.3.The results of CCK-8 detection showed that there was no significant difference in the OD values of RGCs purified and cultured in vitro between different doses of Chinese medicine serum group and the blank group(P>0.05).4.The results of CCK-8 detection showed that the cell survival rate of RGCs purified in vitro and cultured in 3.2mmol/l l L-glutamate for 24 hours was significantly lower than that of the blank group(P<0.05).The cell survival rate of RGCs in medium and high dose Chinese medicine serum intervention group and VB12 intervention group were higher than that of modle group(P<0.05).The cell survival rate of RGCs in high dose Chinese medicine serum intervention group was higher than that of medium dose Chinese medicine serum intervention group and VB12 intervention group(P<0.05).5.The results of Annexin V-FITC/PI flow cytometry showed that the apoptotic rate of RGCs cultured in vitro with 3.2mmol/L L-glutamate for 24hours was significantly higher than that of RGCs in black group(P<0.05),and the apoptotic rate of RGCs in low,medium and high dose Chinese medicine serum intervention group and VB12 intervention group were lower than that of modle group(P<0.05).The apoptosis rate of the intervention group was lower than that of the VB12 intervention group(P<0.05).6.The results of microarray analysis showed that 32 mi RNAs were differentially expressed in model group vs blank group,of which 15 were up-regulated and 17 were down-regulated(P<0.05,|FC|?2).There were 49mi RNAs differentially expressed in the Chinese medicine serum intervention group vs model group,of which 24 were up-regulated and 25 were down-regulated(P<0.05,|FC|?2)and 20 mi RNAs differentially expressed in VB12 intervention group vs model group,among which 11 were up-regulated and 9 were down-regulated(P<0.05,|FC|?2).7.Microarray analysis showed that the expression of mi RNA-132-3p,-222-3p,-29b-3p,-221-3p was down regulated in mode group,but up regulated in Chinese medicine serum intervention group.Only the expression of mi RNA-132-3p and-222-3p was up regulated in VB12 intervention group.8.q RT-PCR was used to verify the expression of mi R-132-3p?-222-3p?-29b-3p?-221-3p.The curve analysis results showed that the amplified products were highly specific and efficient.The results of relative expression calculated by 2-??CTmethod were consistent with those of chip detection.9.The results of target gene prediction showed that the differentially expressed mi RNA may play a role in protecting the optic nerve by regulating the target genes of Slc6a1?Sox5?Melk?Kcna6?Hipk1?Rab1a?Zfp36l2?Fermt2?Cdkn1b?Ddit4?Igf1?Dot1l?Rere?Rarb and Ferr.10.The results of enrichment analysis of GO and KEGG showed that the differentially expressed mi RNA may play a role in protecting the optic nerve by regulating apoptosis,autophagy,immune response,oxidative stress response,heat shock protein,axon,cell proliferation,differentiation and other signaling pathways.Conclusions1.Apoptosis of RGCs in vitro could be induced by L-glutamate with concentration above 0.3mmol/L for more than 12 hours.The cell viability rate of 50%?60%,induced by 3.2 mmol/L L-glutamate for 24 hours,was an ideal RGCs apoptosis model and could be used in the basic research of glaucoma optic nerve protection.2.The Chinese medicine serum could inhibit RGCs apoptosis and enhance RGCs activity in RGCs opoptosis model in vitro induced by L-glutamate,and the effect was concentration dependent.The effect of high Chinese medicine serum was better than that of neurotrophic agent.3.Soothing the liver and unblocking the orifices method might play a role in protecting the optic nerve by regulating the expression of mi RNA-132-3p,-222-3p,-29b-3p and-221-3p.The method up-regulated the expression of these mi RNAs,which was down-regulated when RGCs apoptosis occurred.Compared with the neurotrophic agent VB12,we think that mi RNA-29b-3p and-221-3p might be the specific mi RNA regulating by soothing the liver and unblocking the orifices method.4.The effect of soothing the liver and unblocking the orifices method on optic nerve protection in glaucoma might be achieved by regulating mi RNA expression and its target genes and related downstream pathways.The related target genes included Slc6a1?Sox5?Melk?Kcna6?Hipk1?Rab1a?Zfp36l2?Fermt2?Cdkn1b?Ddit4?Igf1?Dot1l?Rere?Rarb and Fer.The relevant signaling pathways included regulating apoptosis,autophagy,immune response,oxidative stress response,cell proliferation and differentiation. |
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