Protective Effect Of Genipin On Mice With Type 2 Diabetic Nephropathy By Inhibiting Macrophage M1 Polarization

Diabetic nephropathy（DN）is one of the most common and serious complications of type 2 diabetes mellitus（T2DM）and the main cause of end stage renal disease（ESRD）in adults,associated with increased mortality in patients with T2 DM.The pathogenesis of type 2 DN is very complex and still not fully understood,resulting in poor treatment effect.Currently,standard treatments,such as strict control of blood sugar and blood pressure,have been shown to fail to prevent DN from progressing to EDRD,nor to reduce DN-related mortality.Therefore,it is urgent to explore novel therapeutic strategies for DN.Immune and inflammatory responses play important roles in the pathogenesis of DN.The recruitment,accumulation and hyperactivation of pro-inflammatory macrophages in the renal tissue microenvironment are the ultimate drivers of DN inflammation.M1-type（classical）macrophages are pro-inflammatory and M2-type（alternatively activated）macrophages are anti-inflammatory.In the initial stage of infection/injury,M1-type macrophages defend against the invasion of pathogens,and then M2-type macrophages repair the damaged tissue.In the normal microenvironment,there are balanced M1/M2 macrophages to maintain the stability of the human microenvironment.In a hyperglycemic microenvironment,the over-activation of M1 macrophages leads to the imbalance of M1/M2,the increase of mitochondrial reactive oxygen species（ROS）,and the continued aggravation of renal tissue damage.The damaged kidney tissue activates M1-type macrophages again,which eventually leads to irreversible and permanent kidney damage,and even develops into ESRD.Different polarized phenotypes have different metabolic profiles.M1-type macrophages with pro-inflammatory effects mainly rely on glycolytic metabolism;M2-type macrophages with anti-inflammatory effects mainly rely on oxidative phosphorylation.Differences in energy metabolism are the most common metabolic changes that distinguish M1-type macrophages from M2-type macrophages.Gennipin is an active compound isolated from gardenia fruit and an important derivative of aglycones of iridoid glycosides.Gennipin is Normally used as an excellent natural colorant,and it is also an important traditional medicine having the effects of antipyretic、vasodilatory、improved blood flow effects and significant liver protection activity,widely used as anti-inflammatory drugs、anti-angiogenic drugs、antioxidants、hypoglycemic drugs and a variety of inflammation-related diseases.Gennipin can induce the polarization of macrophages M1 towards M2,and its inhibitory effect on inflammation has been widely demonstrated.AS an inhibitor of uncoupling protein 2（UCP2）,gennipin can maintain the balance between ROS production and clearance by regulating the uncoupling function of UCP2,preventing cerebral ischemia reperfusion injury.Therefore,exploring the role of genipin in DN and macrophages may provide new clues for genipin targeting macrophages to reshape the inflammatory microenvironment of DN.In our study,a high-fat diet（HFD）combined with intermittent intraperitoneal injection of low-dose streptozotocin（STZ）was used to construct a type 2 DN mouse model,and genipin was injected intraperitoneally,and the bone marrow-derived macrophages（BMDMs）was obtained to explore the protective effect of genipin on kidneys,and its effects on macrophage phenotype and energy metabolism transformation and mitochondrial ROS production in the DN microenvironment by a series of cell experiments and histological tests,providing a theoretical basis by elucidating the mechanism of genipin’s renal protective effect in DN mouse model and targeting macrophages to remodel the DN inflammatory microenvironment.Methods:（1）The type 2 DN mouse model was constructed in C57BL/6n male mice by HFD combined with multiple low-dose STZ intermittent intraperitoneal injections.Hematoxylin and Eosin（H&E）staining was used to observe renal pathological changes.（2）BMDMs were isolated by cytokine-induced differentiation method of isolated bone marrow monocytes for subsequent experiments.（3）Real-time quantitative polymerase chain reaction was used to detect the basal and Lipopolysaccharide（LPS,1 mg/m L）-induced level of the M1 marker gene of BMDMs in mice,and to explore the influence of macrophages in the type 2 DN mouse model after genipin treatment.（4）Glucose consumption and lactate production were measured by colourimetric method;ATP production was detected by biotin luminescence method;extracellular oxygen consumption rate and acid production rate were detected by oxygen-sensitive and p H-sensitive fluorescent probes to explore the effect of genipin treatment.To explore the regulation of macrophage energy metabolism phenotype in the type 2 DN mouse model after genipin treatment.（5）Mitochondrial ROS production was detected by flow cytometry using MitoSOX^TM reagent.To explore the effect of mitochondrial ROS in macrophages in the type 2 DN mouse model after genipin treatment.Results: HFD combined with multiple low-dose STZ intermittent intraperitoneal injections can successfully build a type 2 DN mouse model.The glycolytic metabolism of BMDMs in type 2 DN mice was enhanced,mitochondrial ROS increased,and M1-type polarization was promoted.After genipin treatment,the glycolytic metabolism of mouse BMDMs decreased,mitochondrial ROS decreased,and M1-type polarization was inhibited.（1）Compared with the con group,the body weight of the mice in the type 2 DN model group did not change significantly,the RBG remained at a high blood sugar level,and there were obvious pathological changes in the kidney tissue,and the treatment of genipin could alleviate the pathological changes in the kidneys.（2）Compared with the con group,with or without LPS induction,M1-type marker genes increased in the BMDMs of mice in the type 2 DN model group,while the expression of M1-type marker genes decreased after genipin treatment.（3）At the basal level,compared with the con group,the glucose uptake of BMDMs in type 2 DN mice was significantly increased after treatment with genipin and type 2DN.Glucose uptake was significantly increased,whereas glucose uptake was significantly decreased after genipin treatment.（4）With or without LPS induction,lactate production was significantly increased in BMDMs of type 2 DN mice compared with the con group,whereas lactate production was significantly decreased after genipin treatment.（5）With or without LPS induction,the ATP levels of BMDMs of type 2 DN mice increased slightly compared with the con group,while the ATP levels of BMDMs of mice were significantly decreased after genipin treatment.（6）With or without LPS induction,the OCR/ECAR ratio of BMDMs in both type2 DN and genipin-treated mice was significantly decreased compared with the con group.However,after genipin treatment,the OCR/ECAR ratio of BMDMs in mice rebounded.（7）Under the induction of LPS,the percentage of mito SOX-positive cells in the BMDMs of mice in the type 2 DN model group increased significantly,and decreased significantly after genipin treatment.Conclusion:In conclusion,this study provides a theoretical support and strong evidence for the clinical application of genipin for type 2 DN by exploring that genipin inhibits their polarization toward M1 macrophages and the production of mitochondrial ROS by regulating the metabolic reprogramming of macrophages,thereby remodeling the inflammatory microenvironment of diabetic nephropathy and reducing kidney damage caused by T2 DM.