| The incidence of diabetic macular edema is high and the degree of visual impairment is serious.At present,retinal laser,anti-vascular endothelial growth factors drugs and glucocorticoids are mainly used in clinical treatment,but single therapy has its corresponding shortcomings.Anti-VEGF drugs as the first-line drugs can effectively reduce the degree of macular edema,but the efficacy is short,need repeated injections and expensive.Traditional Chinese medicine has unique advantages in the treatment of DME.In order to optimize the clinical treatment of DME,we used Buyang Decoction and Conbercept to treat DME.Through clinical and experimental studies,we validated the clinical efficacy of Buyang Decoction and explored its mechanism.Objective:1.To evaluate the efficacy and safety of Buyang Decoction combined with one injection of Conbercept in the treatment of diabetic macular edema(DME),taking three consecutive injections of Conbercept as the control.2.Establish a model of human retinal microvascular endothelial cell(HRMEC)under high glucose to lay a foundation for exploring the mechanism of Buyang Decoction.3.To observe the effects of serum containing Buyang Decoction on the proliferation of human retinal microvascular endothelial cells,vascular endothelial growth factor(VEGF)and tight junction(TJ)protein occludin under high glucose,and to explore its mechanism of protecting blood-retinal barrier(i-BRB).To provide experimental basis for the treatment of diabetic macular edema with Buyang Decoction.Method:1.Clinical research methods:135 patients with diabetic macular edema who met the exclusion criteria were randomly divided into two groups according to the allocation ratio of 2:1 according to the prospective,non-inferior design principle and multi-center,randomized and controlled methods.The treatment group was given Chinese herbal medicine orally for 4 months,combined with the first-month intravitreal injection of Conbercept once;the control group was given intravitreal injection of Conbercept three times,once a month.The two groups were followed up for 3 months.The best corrected visual acuity(BCVA),central retinal thickness(CRT),TCM syndrome score,laboratory examination,ocular signs and combined medication were recorded before and after treatment.BCVA and CRT were taken as the main evaluation indexes.Liver and kidney function test values were used as safety indicators to evaluate the efficacy and safety of Buyang Decoction combined with single injection of Conbercept in the treatment of DME.2.Experimental research methods:(1)Human retinal microvascular endothelial cells(HRMEC)were treated with DMEM complete medium with 25 mol/L glucose concentration.The viability of HRMEC cells was measured by CCK8 method after 24 and 48 hours of culture,respectively,and a model of human retinal microvascular endothelial cells was established under high glucose.(2)Buyang decoction-containing serum and blank control serum of SD rats were prepared.CCK8 method was used to detect the effect of serum containing Buyang Decoction on the proliferation of human retinal microvascular endothelial cells under high glucose.(3)Establish a model of human retinal microvascular endothelial cells under high glucose.Low,medium and high concentration of Buyang decoction-containing serum and blank control serum interfere with human retinal microvascular endothelial cells under high glucose and normal glucose respectively.RT-PCR was used to detect the mRNA expression of VEGF and occludin in HRMEC cells,and Western-blot was used to detect the relative expression of VEGF and occludin protein in HRMEC cells.Result:1.Clinical research(1)The changes of main therapeutic indexes compared with baseline:BCVA in the experimental group changed by 5.46±7.01 in one month,4.7.1±9.62 in two months,2.51 ±8.48 in three months,1.91± 9.83 in four months and 4.05±6.22 in seven months.BCVA in the control group changed 3.66±9.42 in one month,4.56±8.77 in two months,6.23±6.58 in three months,3.08.1±7.57 in four months,and 1.44±7.07 in long-term follow-up in seven months.The CRT of the experimental group was followed up for one month with baseline change of-132.71±121.82?m,for two months with baseline change of-75.69± 134.25?m,for three months with baseline change of-33.04±129.0?m,for four months with baseline change of-61.7±129.79?m,and for seven months with long-term follow-up with baseline change of-39.94 ± 144.88?m.The CRT of the control group was followed up for one month with baseline change-135.67± 137.56?m and for two months with baseline change-175.84±139.76?m and for three months with baseline change-185.19±130.29?m and for four months with baseline change-129.85± 155.18?m and for seven months with long-term follow-up with baseline change-69.89.1±127.83?m,(2)Treatment outcome judgment of main therapeutic indicators:there were 3 cases(3.75%)of macular edema decrease degree more than or equal to 50%of baseline in the experimental group,50 cases(62.5%)of macular edema decrease degree more than or equal to 25%and less than 50%,27 cases(33.75%)of no change or aggravated macular edema degree;there were 4 cases(9.7%)of macular edema decrease degree more than or equal to 50%of baseline in the control group,27 cases(65.85%)of macular edema decrease degree more than or equal to 25%and less than 50%in the control group.There were 10 cases(24.39%)with no change or aggravation of edema in the control group.There were 13 cases(16.25%)with the best corrected visual acuity(?10 letters),36 cases(45%)with the best corrected visual acuity(?5 letters and<10letters)and 31 cases(38.75%)with no change or visual impairment in the experimental group;there were 8 cases(19.51%)with the best corrected visual acuity(?10 letters)in the control group,and 15 cases(36.58%)with the best corrected visual acuity(?5 letters and<10 letters)in the control group.A total of 18 cases(43.9%)declined in the control group.The total effective rate of the experimental group was 15 markedly effective cases(18.75%),47 effective cases(58.75%)and 18 invalid cases(22.5%).The total effective rate of the control group was 11 markedly effective cases(26.83%),24 effective cases(58.54%)and 6 invalid cases(14.63%).(3)Non-inferiority test results of main efficacy indicators:the total effective rate difference between the experimental group and the control group was-7.86%,Cu=8.11%,lower than the non-inferiority threshold value of 20%,rejecting the invalid hypothesis,the total effective rate of TCM treatment group was not inferior to the positive drug control group,and the non-inferiority test was established;the BCVA effective rate difference between the experimental group and the control group was 6.07%,Cu=13.41%,lower than the non-inferiority threshold value of 20%,and refused absolutely ineffective hypothesis,on BCVA effective ratio Chinese medicine combined treatment group was not inferior to the positive drug control group,non-inferior test was established;the difference of CRT effective rate between the experimental group and the control group was-9.35%,Cu=7.39%,lower than the non-inferior threshold value of 20%,rejected the invalid hypothesis,on CRT effective ratio Chinese medicine combined treatment group was not inferior to the positive drug control group,non-inferior test was established.2.Experimental research(1)The effect of serum containing Buyang Decoction on cell proliferation:the proliferation coeff-icient of HRMECs with high glucose 25 mmol/L was higher than that with low concentration 5 mmol/L.The proliferation coefficient of HRMECs in the experimental group was significantly lower than that in the model control group(P=0.025),but the difference between the low dose experimental group and the normal control group was small at 24h,and the proliferation coefficient was close(P=0.19).(2)The effect of serum containing Buyang Decoction on the expression of vascular endothelial growth factor in HRMEC cells:The mRNA expression of vascular endothelial growth factor in model control group was significantly higher than that in normal control group(P=0.000);the relative expression of target genes of vascular endothelial growth factor in all experimental groups was lower than that in model control group(P<0.05);the medium dose experimental group had the greatest influence on the expression of vascular endothelial growth factor.The protein expression of vascular endothelial growth factor in normal control group was lower than that in model control group(P=0.037).The protein expression of vascular endothelial growth factor in middle-dose and high-dose drug-containing serum group decreased,especially in medium-dose drug-containing serum group.There was significant difference between the two groups(P<0.05).(3)The effect of serum containing Buyang Decoction on occludin expression in HRMEC cells:The mRNA expression of occludin protein in normal control group was higher than that in model control group,and there was significant difference between groups(P=0.033).The mRNA expression of occludin protein was increased in all experimental groups.Compared with model group,the mRNA expression of occludin protein in low dose group and middle dose group increased significantly(P<0.05).There was little difference between high dose serum group and model group,and there was no statistical difference between the two groups(P=0.156).The relative protein expression of occludin in normal control group was significantly higher than that in model group(P=0.000).The protein expression of occludin in all experimental groups was higher than that in model group.The relative expression in low dose experimental group was the highest,and there was significant difference between two groups(P<0.05).Conclusion:1.The efficacy of Buyang Decoction plus Conbercept single injection in the treatment of DME was not inferior to that of Coonbercept three consecutive injections treatment,which can improve the best corrected visual acuity,alleviate macular edema and improve the overall symptoms of patients.The joint scheme was economical,safe and reliable.2.High glucose(25 mmol/L)can induce human retinal miacrovascular endothelial cell proliferation,increase the expression of vascular endothelial growth factors,decrease the expression of tight junction protein occludin,and disturb cell function.25 mmol/L sugar concentration can establish a stable experimental model of high glucose human retinal microvascular endothelial cells.3.The serum containing Buyang Decoction may interfere with the pathogenesis of DME by inhibiting the abnormal proliferation caused by high glucose environment;it may protect the function of blood-retinal barrier by reducing the expression of vascular endothelial growth factor and increasing the expression of occludin protein. |
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