Protective Effects And Mechanism Investigation Of Honokiol On Diabetic Peripheral Neuropathy

Diabetic peripheral neuropathy（DPN）is one of the microvascular complications of diabetes with complex pathogenesis and high morbidity.Honokiol（HNK）,a natural product mainly extracted from Magnolia officinalis,has been shown to have various pharmacological activities against different diseases,but the current role and mechanism of Honokiol on diabetic peripheral neuropathy.Not yet researched and reported.Therefore,this study aimed to study the protective effect of honokiol on the rat Schwann cell line RSC96 cells damaged by high-dose glucose of 150 m M,and the protective effect of honokiol on streptozotocin（STZ）-induced diabetes mellitus.The therapeutic effect of diabetic peripheral neuropathy in mice,and its potential protective mechanism was further studied and discussed.In vivo,Sprague-Dawley（SD）rats were injected intraperitoneally with streptozotocin(60 mg·kg^-1)to establish DPN rats,and were orally administered different doses of HNK(25,50,100 mg·kg^-1·d^-1)for 8 weeks treatment,oral alpha-lipoic acid(ALA,100 mg·kg^-1·d^-1)was used as a positive control.The changes of body weight and blood sugar of the rats were monitored,the thermal pain and mechanical pain threshold changes of the rats were detected,the conduction velocity of the sciatic nerve was recorded,and the serum oxidative stress indexes of the rats were detected.The ultrastructural changes of sciatic nerve in DPN rats were observed by electron microscope.The results showed that rat HNK could significantly increase the nerve conduction velocity,improve the thermal pain,mechanical pain threshold,and the ultrastructural morphology of the sciatic nerve in DPN rats,and reduce the level of oxidative stress in vivo.In vitro,RSC96 cells were treated with 150 m M glucose and different concentrations（2.5,5,10μM）of HNK for 48 h to establish an in vitro DPN cell model.Cell viability was assessed by MTT assay.In addition,neural function was assessed by measuring nerve growth factor（NGF）secretion capacity in RSC96 cells by ELISA.Antioxidative effects were assessed by reactive oxygen species（ROS）assay,malondialdehyde（MDA）assay,and glutathione（GSH）assay.Mitochondrial function was examined by mitochondrial membrane potential（MMP）assay,ATP content assay,and mitochondrial DNA（mt DNA）copy number was detected by fluorescence quantitative PCR.Fe²⁺assays were used to assess the effect of HNK on ferroptosis.The m RNA and protein expression of the genes were analyzed by RT-q PCR and Western blotting.The results show that at the cellular level,HNK can enhance cell viability and increase the secretion capacity of Schwann cells NGF.HNK exhibited powerful antioxidant effects by eliminating ROS,MDA and increasing GSH content.At the same time,HNK treatment ameliorated the reduction and decrease of mitochondrial membrane potential,ATP production,and mt DNA copy number caused by high glucose injury.HNK treatment also decreased cellular iron levels and improved cellular ferroptosis.Mechanistically,HNK treatment promoted the expression of SIRT1,p-AMPK,PGC-1α,NRF2,and NGF,and regulated hyperglycemia in vivo and in vitro with antioxidants（HO-1,SOD1,SOD2,and KEAP1）,m RNA and protein expression of genes related to mitochondrial function（TFAM）and ferroptosis（GPX4,SLC7A11,FTH-1,and Tf R1）.In the presence of the specific SIRT1 inhibitor EX527,it was further confirmed that honokiol exerts neuroprotective effects by activating SIRT1.In conclusion,the results of this study indicate that Honokiol can improve oxidative stress,regulate mitochondrial function and ferroptosis by activating the SIRT1/NRF2/PGC-1αsignaling pathway and regulating downstream genes,thereby improving experimental diabetic peripheral neuropathy.This study provides a reference for the prevention and treatment of honokiol in diabetic peripheral neuropathy.