Study On The Ameliorative Effect Of Atractylodis Rhizoma In Fructose-induced Rat Glomerular Podocyte Injury Through Antioxidant And Its Molecular Mechanism

Atractylodis rhizome（AR）is a traditional Chinese medicine for aromatic damp-resolving.AR and its active ingredient have anti-oxidation and anti-inflammatory pharmacological effects.Ermiao Pill with AR as the primal medicine are often used to treat syndrome of dampness-heat in lower jiao and have the kidney protective effects,but the kidney protective mechanism of AR still needs to be elucidated.In this paper,we will analyze the components of Atractylodis Rhizoma water extract（ARE）and conduct network pharmacological research preliminary to find the possible pathways and component clues of AR kidney protection,and explore possible molecular mechanisms from the perspective of glomerular podocyte protection.In this paper,ARE was sequentially extracted with petroleum ether,ethyl acetate,and n-butanol.Liquid chromatography-tandem mass spectrometry（LC-MS/MS）analysis of each extracted phase and the remaining aqueous phase,combined with the analysis results of high performance liquid chromatography（HPLC）,initially determined that ARE may contain 36 components including atractylodin.Based on the analysis results of AR components,network pharmacology research was performed,and the predicted results suggested that AR may ameliorate renal oxidative stress.The results of clinical and experimental researches show that long-term high fructose diets could induce kidney dysfunction.Our previous study observed oxidative stress such as high-level hydrogen peroxide（H₂O₂）and superoxide anion（O₂^-）,glomerular podocyte processes effacement and proteinuria in fructose-fed rats.Reactive oxygen species（ROS）and H₂O₂ could activate transient receptor potential cation channel 6（TRPC6）,which leads to the up-regulation of intracellular Ca²⁺levels.Synaptopodin,an important molecule in podocyte slit diaphragm,inhibits over-migration of podocyte by regulating the small G protein family Ras-related C3botulinum toxin substrate（Rho A）and cell division control protein 42（Cdc42）to stabilize podocyte stress fibers and inhibit the formation of filamentous pseudopods.Unbalanced motility of podocyte will promote the podocyte processes effacement and the destruction of the slit diaphragm.Some scholars have observed that the high expression of calcium/calmodulin-dependent protein kinase 4（CaMK4）promotes the degradation of Synaptopodin and exacerbates podocyte migration in the adriamycin-induced animal nephropathy model and lipopolysaccharide（LPS）-induced podocyte injury model.Therefore,this paper will investigate whether fructose-induced oxidative stress promotes podocyte migration?And the role of TRPC6-Ca²⁺-CaMK4-Synaptopodin signaling in this process.This thesis focuses on the study of the ARE to reduce injury of glomerular podocytes based on the 10%fructose drinking water-fed rat nephropathy model established by us previously.After 9 weeks of oral administration of ARE（720 and1090 mg/kg）or allopurinol（5 mg/kg）to model rats,improved insulin resistance,reduced serum uric acid,creatinine,urea nitrogen levels and urine albumin/creatinine ratio.At the same time,ARE and allopurinol reduced the levels of H₂O₂ and malondialdehyde（MDA）,and enhance superoxide dismutase（SOD）activity in the renal cortex of model rats,and increased the protein levels of glomerular catalase（CAT）and SOD1,which is consistent with the results of inhibiting glomerular basement membrane thickening and podocyte foot processes effacement.Further research found that ARE and allopurinol could significantly down-regulate the expression of TRPC6and p-CaMK4 in the glomerulus of fructose-model rats,and increase the protein level of Synaptopodin.These findings suggest that ARE inhibits high fructose-induced oxidative stress and improves glomerular podocyte damage,and its protective mechanism may be related to the regulation of TRPC6-CaMK4-Synaptopodin signaling.Atractylodin,the main component of AR,has antioxidant effects.This article will further investigate whether atractylodin could inhibit fructose-induced podocytes over-migration through anti-oxidant.Using 5 m M fructose to stimulate podocytes cultured in vitro（24 h）,and enhanced podocyte migration was observed.The intervention of Atractylodin（20,40 and 80μM）or allopurinol（100μΜ）reduced the excessive migration of podocytes and intracellular ROS levels,restored the expression of F-actin in podocytes,and increased the protein levels of podocin,nephrin,α-Actinin-4,CD2AP and Synaptopodin,down-regulated the expression of TRPC6 and p-CaMK4 in the fructose model group.These findings suggest that atractylodin may inhibit fructose-induced oxidative stress to prevent podocyte over-migration and reduce podocyte damage,and its protective mechanism may be related to the regulation of calcium signaling.To further investigate whether fructose promotes excessive migration of podocytes through oxidative stress and the regulatory role of TRPC6 and CaMK4 in this process.We intervened podocytes with the CaMK4 si RNA,TRPC6 si RNA and antioxidant N-Acetyl-L-cysteine（NAC）,respectively.The results showed that CaMK4 si RNA,and CaMK4 si RNA combined with atractylodin or allopurinol could increase the protein level of Synaptopodin and inhibit over-migration of podocytes in the model group;After using TRPC6 si RNA and TRPC6 si RNA combinated with atractylodin or allopurinol,down-regulated intracellular Ca²⁺and the protein level of p-CaMK4,restored the expression of Synaptopodin,and effectively inhibited the over-migration of podocyte in the model group,suggesting that fructose activates TRPC6 channel to induce Ca²⁺influx,phosphorylating CaMK4 promotes the degradation of Synaptopodin,thereby promoting podocyte migration.NAC,and NAC combined with atractylodin or allopurinol inhibited the over-migration of podocyte,down-regulated the expression of TRPC6 and p-CaMK4,and reduced intracellular Ca²⁺levels in model group,suggesting that fructose promotes podocyte migration through oxidative stress to activate TRPC6channel.The above results indicate that fructose induces podocyte migration through oxidative stress to regulate TRPC6-Ca²⁺-CaMK4-Synaptopodin signaling,and atractylodin might reduce fructose-induced podocyte over-migration through anti-oxidant to regulate TRPC6-Ca²⁺-CaMK4-Synaptopodin signaling.Conclusion:In this paper,we found a new way of fructose-induced glomerular podocyte damage:Activation of TRPC6-CaMK4-Synaptopodin signaling by oxidative stress to induce glomerular podocytes damage.ARE could improve fructose-induced rat glomerular podocyte damage,and its protective mechanism may through anti-oxidant to regulate TRPC6-CaMK4-Synaptopodin signaling.Further research found the active ingredient atractylodin of AR might inhibit fructose-induced over-migration of podocyte through anti-oxidant to regulate TRPC6-Ca²⁺-CaMK4-Synaptopodin signaling.These works provide an experimental basis for the clinical prevention and treatment of kidney damage caused by fructose-diet.