杞菊地黄丸基于NLRP3/Caspase-1/GSDMD信号通路治疗糖尿病视网膜病变的作用机制OA

Objective:To investigate the protective effect of Qiju Dihuang Wan on diabetic retinopathy(DR)and its regulatory mechanism on the NOD-like receptor protein 3/Cysteinyl aspartate-specific proteinase-1/Gasdermin D(NLRP3/Caspase-1/GSDMD)signaling pathway.Methods:Human retinal microvascular endothelial cells(HRMEC)were divided into the blank group,model group,and Qiju Dihuang Wan low-and high-dose groups.Except for the blank group,all groups were induced with 40 mmol·L-1 high glucose,and the treatment groups were supplemented with 6%and 12%Qiju Dihuang Wan-containing serum,respectively,based on the induction.After 24 h of culture,the levels of interleukin(IL)-1β and IL-18 were measured by enzyme-linked immunosorbent assay(ELISA),pyroptosis-related protein expression was detected by immunofluorescence and Western blot,and the mRNA levels of NLRP3/Caspase-1/GSDMD signaling pathway-related molecules were determined by real-time polymerase chain reaction(Real-time PCR).The mice were randomly divided into the blank group,model group,Qiju Dihuang Wan low-,medium-,and high-dose groups(11,22,44 g·kg-1·d-1),and calcium dobesilate group(0.13 g·kg-1·d-1).Except for the blank group,all groups were intraperitoneally injected with streptozotocin to establish the DR model.After successful modeling,the treatment groups received corresponding doses of Qiju Dihuang Wan crude drug or calcium dobesilate suspension by gavage once daily,while the blank group and model group received an equal volume of normal saline,for 8 consecutive weeks.Fundus changes were examined by optical coherence tomography,optical coherence tomography angiography,and fundus fluorescein angiography.Retinal pathological morphology was observed by hematoxylin-eosin staining.Real-time PCR was used to detect the mRNA levels of NLRP3,Caspase-1,GSDMD,IL-1β,and IL-18 in retinal tissue,and Western blot was used to detect the protein expression of NLRP3,ASC,cleaved Caspase-1,and GSDMD-N.Results:Cell experiments showed that compared with the blank group,the model group showed significantly decreased HRMEC viability(P<0.01),and significantly increased levels of IL-1β,IL-18,as well as increased protein expression of NLRP3,cleaved Caspase-1,and GSDMD-N(P<0.01).Compared with the model group,the above indicators were significantly reduced in the high-dose Qiju Dihuang Wan groups(P<0.05,P<0.01).Animal experiments showed that compared with the blank group,the model group had elevated blood glucose(P<0.01),decreased retinal thickness and vascular density(P<0.01),up-regulated mRNA expression of NLRP3,Caspase-1,GSDMD,IL-1β and IL-18 in retinal tissue(P<0.01),and up-regulated protein expression of NLRP3,ASC,cleaved Caspase-1 and GSDMD-N(P<0.01).Compared with the model group,the Qiju Dihuang Wan medium-and high-dose groups showed decreased blood glucose(P<0.01),increased retinal thickness and vascular density(P<0.01),alleviated retinal pathological damage,and down-regulated mRNA expression of NLRP3,Caspase-1,GSDMD,IL-1β,IL-18,as well as protein expression of NLRP3,ASC,cleaved Caspase-1,and GSDMD-N(P<0.05,P<0.01).Conclusion:Qiju Dihuang Wan may exert protective effects against diabetic retinopathy by inhibiting the activation of the NLRP3/Caspase-1/GSDMD signaling pathway and alleviating high glucose-induced retinal pyroptosis.