Protective Effect Of Danggui Buxue Decoction Against Hypoxia-induced Damage Of Retinal Müller Cells And The Molecula Mechanism Research

Objective:Diabetic retinopathy(DR)is one of the most common microvascular complications in diabetes.Early diabetic retinopathy includes neuropathy and vascular disease,and neuropathy may precede and aggravate the occurrence of vascular disease.Most treatments target vascular lesions with limited efficacy.From the perspective of the pathogenesis of "qi and blood deficiency,blood stasis and collaterals",we found that Danggui Buxue Decoction(RRP)can effectively alleviate the vascular lesions of early diabetic retina.Based on the "multi-targeting,multi-mechanism"combination characteristics,combined with previous in vivo experiments,RRP can alleviate the abnormal activation of diabetic retinal glial cells(Miiller cells).This study aimed to explore the effect of Danggui Buxue Decoction(RRP)on the damage of glial cells(Muller cells)in diabetic retina and its related molecular mechanisms.Methods:The human retinal Muller cell line MIO-M1 was used to stimulate MIO-M1 to establish a damage model with high glucose and hypoxia,and to explore cell survival levels(cell viability,apoptosis,proliferation)and related angiogenesis factors and inflammatory factors by high glucose and hypoxia.Rats were continuously intragastrically administrated with Danggui Buxue Decoction(RRP)for 7 days to obtain drug-containing serum.After inactivation,the drug-containing serum was added to the serum-free medium at a ratio of 10%to intervene the cells;Cell viability was detected by CCK8 assay,apoptosis was detected by Annexin V-FITC apoptosis assay kit,cell proliferation ability was detected by EdU kit,mRNA expression of angiogenic factor and inflammatory factor was detected by qRT-PCR.The expression of endoplasmic reticulum stress(ER stress)-related protein was detected by Western blot.Results:1.After hypoxia(1%O2)stimulation for 48 hr,72 hr,cell viability decreased;while high glucose(25 mmol/L)stimulation had no significant effect on cell viability,high glucose combined with hypoxia had no further damage to cells;2.After 48 hours of hypoxia(1%02)stimulation,the cell proliferation ability decreased;similarly,high glucose(25mmol/L)stimulation had no significant effect on cell proliferation ability,and high glucose combined with hypoxia did not further damage the cells;3.After 48 hours of hypoxia stimulation,MIO-M1 increased the level of apoptosis;4.After 48 hours of hypoxia stimulation,MIO-M1 angiogenesis factor(ANGPTL4,VEGF)and inflammatory factors(IL-1?,ICAM-1)mRNA expression up-regulation;5.Danggui Buxue Decoction(RRP)had no significant effect on cell viability and proliferation of MIO-M1 under hypoxia.6.Danggui Buxue Decoction(RRP)can significantly alleviate hypoxia-induced apoptosis of MIO-M1 cells.7.Danggui Buxue Decoction(RRP)did not affect the expression of MIO-M1 angiogenesis factor ANGPTL4 and VEGF induced by hypoxia stimulation,but it could relieve the up-regulation of inflammatory factors IL-1? and ICAM-1 to some extent.8.After adding endoplasmic reticulum stress agonist TM(tunicamycin),the level of apoptosis increased;After adding the endoplasmic reticulum stress inhibitor TUDCA(taursinoxydeoxycholic acid),the level of apoptosis did not rise after hypoxia.9.Danggui Buxue Decoction(RRP)can alleviate the activation of endoplasmic reticulum stress ATF4/CHOP pathway in M10-M1 cells induced by hypoxia.Conclusion:By comparing the pathological microenvironment of high glucose and hypoxia in the pathogenesis of diabetic retinopathy,we found that:Retinal Muller cells are more sensitive to hypoxia(1%02)stimulation;Hypoxia(1%02)can cause decreased cell viability and proliferative capacity,increased apoptosis,and increased expression of angiogenic and proinflammatory factors in retinal Muller cells;Danggui Buxue Decoction(RRP)can effectively alleviate the increase of apoptosis induced by hypoxia(1%O2).To alleviate the up-regulation of inflammatory factor ICAM-1,but no significant impro.vement on angio gsenic factors;RRP may attenuate hypoxia-induced apoptosis in retinal Muller cells by inhibiting ATF4/CHOP pathway in endoplasmic reticulum stress.This study suggests that Miiller apoptosis and activation of ATF4/CHOP may be one of the targets and mechanisms of RRP in relieving diabetic retinopathy.