Effects Of Tangwang Huayu Oral Liquid On DME Patients Due To Deficiency Of Yin And Yang Syndrome And Inhibitory Effects Of Hirudo On Fibroblast Proliferation And Its Related Mechanism

Purpose OCTA was used to evaluate the macular perfusion of patients due to Deficiency of Yin and Yang Syndrome with diabetic macular edema treated by Tangwang Huayu Oral Liquid.To investigate the effect of hirudo on fibroblasts proliferation and its molecular mechanism.Methods 1.Thirty-six diabetic eyes with diabetic macular edema,thirty-nine diabetic eyes without macular edema and thirty-eight eyes of healthy controls were examined using OCTA（Heidelberg Engineering,Heidelberg,Germany）and OCT.The macular vessel area density（VD）,foveal avascular zone（FAZ）area,numbers of MAs,best corrected visual acuity（BCVA）and central mcular thickness（CMT）were assessed and statistically compared among the three groups.2.Sixty-five patients were randomly divided into PRP group,oral liquid group（Tangwang Huayu Oral Liquid）and combination group（PRP and Tangwang Huayu Oral Liquid）underwent a six-month course of treatment,and the changes of Logarithm of the minimum angle of resolution（Log MAR）,central mcular thickness（CMT）,foveal avascular zone（FAZ）,vessel area density（VD）were detected before and 1,3,6 months after treatment.15 patients due to Deficiency of Yin and Yang Syndrome with diabetic macular edema were collected and scanned by thermal texture maps（TTM）before and 15 days after treating by Tangwang Huayu Oral Liquid.3.CCK-8 test,cell counting,Edu and TUNEL incorporation assay was used to observe the effect of hirudo on the proliferation and apoptosis of NIH3T3 cells,RT-PCR was used to explore the effect of hirudo on the relative expression of p38,TAK1,TGF-β and caspase-3 genes.Western blot was used to investigated the effect of hirudo on the relative expression of TGF-β,TAK1,p38,p-p38 and caspase-3 protrins.Results 1.OCTA was used to evaluate macular perfusion in patients with diabetic macular edema.（1）.general data:All patients were diagnosed as type 2 diabetes（mean age 59.413±2.721 years;course of diabetes 8.8±0.57 years;Hb A1 c 7.744±1.241%）.（2）.FAZ: Compared with patients in control group and NPDR without DME group,DME patients had significantly larger FAZ area（both SVC and DVC）（p<0.05*）.NPDR without DME patients had significantly larger FAZ area at the deep vascular complex than that at the superficial vascular complex（p=0.041#）.（3）.VD: Compared with patients in control group and NPDR without DME group,DME patients had significantly lower VD（both SVC and DVC）（p<0.05*）.DME and NPDR without DME patients had significantly lower VD at the deep vascular complex than that at the superficial vascular complex（p=0.019#,p=0.037#）.（4）.MAs: Compared with patients in control group and NPDR without DME group,DME patients had significantly more MAs（both SVC and DVC）（p<0.05*）.DME and NPDR without DME patients had significantly more MAs at the deep vascular complex than that at the superficial vascular complex（p=0.027#,p=0.001##）.（5）.Syndrome: The incidence of deficiency of Yin and Yang syndrome in DME group was higher than that in NPDR without DME and control group.2.The macular perfusion of patients due to Deficiency of Yin and Yang Syndrome with diabetic macular edema treated by Tangwang Huayu Oral Liquid according to OCTA.（1）.general data:All patients were diagnosed as type 2 diabetes（mean age 58.036±9.448 years;course of diabetes 8.782±0.582 years;Hb A1 c 6.918±0.857%）.（2）.FAZ: There was no statistical difference in FAZ before and after treatment among the three groups according to OCTA（p> 0.05）.（3）.VD: There was no statistical difference in VD before and after treatment among the three groups according to OCTA（p> 0.05）.（4）.BCVA: There was no statistical difference in BCVA before and one month after treatment in PRP group and combination group.The BCVA of the three groups were significantly improved three and six month after treatment（p< 0.05）.（5）.CMT: The CMT of PRP group was significantly increased one month after treatment.The CMT of the three groups were significantly reduced three and six month after treatment,especially for the 6^th month（p< 0.05）.（6）.TTM: The △T of eyes were reduced（p=0.001**）,and the temperature of forehead,face and upper part of head were increased（p < 0.05）.3.The effect of hirudo on fibroblasts proliferation and its molecular mechanism.（1）.Conditions: Hirudo with a concentration of 0.006U/ml and the 5th day were selected to explore the proliferation of NIH3T3 cells.（2）.Proliferation inhibition: The number of NIH3T3 cells decreased according to cell counting（p=0.003**）,the OD value decreased by CCK-8（p=0.006**）and the positive cell rate of Edu（p=0.007**）decreased compared with the control group 5 days after hirudo intervention.（3）.Apoptosis: The positive cell rate of TUNEL（p=0.004**）increased compared with the control group 5 days after hirudo intervention.（4）.p38 MAPK signaling pathway: According to the RT-PCR and Western blot,the gene and protein relative expression of p38（p=0.000***,p=0.000***）,TAK1（p=0.020*,p=0.005**）,TGF-β（p=0.030*,p=0.003**）decreased 5 days after hirudo intervention.（5）.caspase-3 pathway: According to the RT-PCR and Western blot,the gene and protein relative expression of caspase-3（p=0.043*,p=0.000***）increased 5 days after hirudo intervention.Conclusion 1.The macular perfusion of DME patients were decreased according to OCTA.2.For patients of DME due to Deficiency of yin and Yang Syndrome,Tangwang Huayu Oral Liquid can maintain their macular perfusion in 6 months.3.Hirudo was effective in inhibiting NIH3T3 proliferation of by inhibiting TGF-β/TAK/p-38 signaling pathway.