ABSTRACT
ObjectiveTo investigate the mechanism of Buyang Huanwutang in treating diabetic peripheral neuropathy （DPN） via mitochondrial transport. MethodDiabetes in SD rats was induced by a high-carbohydrate/high-fat diet and intraperitoneal injection of streptozotocin （STZ）. The 45 diabetic rats were randomly assigned into a DPN group， an alpha-lipoic acid （60 mg·kg-1·d-1） group， and a Buyang Huanwutang （15 g·kg-1·d-1） group， with 15 rats in each group. Fifteen normal SD rats were fed with the standard diet and set as the control group. The rats were administrated with corresponding drugs by gavage for 12 weeks. The paw withdraw threshold （PWT） and motor nerve conduction velocity （MNCV） were measured at the end of medication， and the sciatic nerve and the bilateral dorsal root ganglia of L4-5 were collected. The injury model of NSC34 cells was established by treating with 50 mmol·L-1 glucose and 250 μmol·L-1 sodium palmitate. The NSC34 cells were then randomly assigned into a blank （10% blank serum） group， a DPN （10% blank serum） group， an apha-lipoic acid （10% apha-lipoic acid-containing serum） group， a Buyang Huanwutang （10% Buyang Huanwutang-containing serum） group， and a Buyang Huanwutang + Compound C （CC） （10% Buyang Huanwutang-containing serum + 10 μmol·L-1 CC） group. The cell intervention lasted for 24 h. The immunofluorescence method， immunohistochemistry， and Western blot were employed to determine the expression levels of phosphorylated adenosine monophosphate-activated protein kinase （p-AMPK）， phosphorylated cAMP-response element binding protein （p-CREB）， kinesin family member 5A （KIF5A）， and dynein cytoplasmic 1 intermediate chain 2 （DYNC1I2）. ResultCompared with the control group， the DPN group of rats showed increased fasting blood glucose （P<0.01）， decreased MNCV and PWT （P<0.01）， down-regulated expression of KIF5A， p-AMPK/AMPK， and p-CREB/CREB （P<0.01）， and up-regulated expression of DYNC1I2 （P<0.01）. Compared with the DPN group， drug intervention groups showed increased MNCV and PWT （P<0.01）， up-regulated expression of KIF5A， p-AMPK/AMPK， and p-CREB/CREB （P<0.05， P<0.01）， and down-regulated expression of DYNC1I2 （P<0.05， P<0.01）. The Buyang Huanwutang group had higher levels of MNCV and KIF5A （P<0.05） and lower level of DYNC1I2 （P<0.01） than the apha-lipoic acid group. Compared with the blank group， the DPN group of NSC34 cells showed decreased levels of KIF5A， p-AMPK/AMPK， and p-CREB/CREB （P<0.01） and increased level of DYNC1I2 （P<0.01）. The apha-lipoic acid group and Buyang Huanwutang group had higher levels of KIF5A， p-AMPK/AMPK， and p-CREB/CREB （P<0.05， P<0.01） and lower level of DYNC1I2 （P<0.01） in NSC34 cells than the DPN group. Buyang Huanwutang group had higher KIF5A level （P<0.05） in NSC34 cells than the apha-lipoic acid group. Moreover， the Buyang Huanwutang + CC group had lower levels of KIF5A， DYNC1I2， p-AMPK/AMPK， and p-CREB/CREB （P<0.01） in NSC34 cells than the Buyang Huanwutang group. ConclusionBuyang Huanwutang may regulate mitochondrial anterograde transport via the AMPK/CREB pathway to prevent and treat DPN.
ABSTRACT
ObjectiveTo explore the neuroprotective mechanism of Buyang Huanwutang （BYHW） on diabetic peripheral neuropathy （DPN） rats based on oxidative stress and investigate the dosage of Astragali Radix （AR）. MethodNinety SD rats were randomly divided into a normal group， a model group， an α-lipoic acid group （60 mg·kg-1·d-1）， and BYHW groups with high- （15 g·kg-1·d-1）， medium- （8.75 g·kg-1·d-1）， and low-dose （5.625 g·kg-1·d-1） AR groups. The diabetes model was induced in rats except for those in the normal group by the high-sugar/high-fat diet and intraperitoneal injection of streptozotocin （STZ）. Drug intervention lasted for 12 weeks. The paw withdrawal threshold （PWT） and sensory nerve conduction velocity （SNCV） were detected after drug intervention. Gonad-stimulating hormone （GSH） and malondialdehyde （MDA） were determined. The mitochondrial morphology and structure in sensory neurons of L4-5 dorsal root ganglion （DRG） of rats were observed by electron microscopy. Respiratory chain complex Ⅰ， Ⅱ， Ⅲ， and Ⅳ activities and the mitochondrial membrane potential were detected. The main proteins in the adenosine monophosphate-activated protein kinase （AMPK）/nuclear factor-related factor-2 （Nrf2） pathway， such as phosphorylated AMPK （p-AMPK）， phosphorylated Nrf2（p-Nrf2）， heme oxygenase-1 （HO-1）， and quinone NADH dehydrogenase 1 （NQO1）， were detected by immunohistochemistry and Western blot. ResultCompared with the normal group， the model group showed increased fasting blood glucose （P<0.01）， decreased content of SNCV， PWT， and GSH （P<0.01）， elevated MDA content （P<0.01）， obvious mitochondrial damage with vacuolations， reduced activities of respiratory chain complex Ⅰ， Ⅱ， Ⅲ， and Ⅳ and mitochondrial membrane potential （P<0.01）， and declining p-AMPK， p-Nrf2， HO-1， and NQO1 （P<0.01）. Compared with the model group， the α-lipoic acid group and BYHW high-dose group showed increased SNCV， PWT， and GSH， decreased MDA （P<0.05， P<0.01）， alleviated mitochondrial structural damage， increased respiratory chain complex Ⅰ， Ⅱ， Ⅲ， and Ⅳ activities and mitochondrial membrane potential （P<0.01）， and elevated p-AMPK， p-Nrf2， HO-1， and NQO1 （P<0.05， P<0.01）. ConclusionBYHW regulates oxidative stress through the AMPK/Nrf2 pathway to treat DPN. The therapeutic effect of BYHW is related to the dosage of AR. The BYHW group with high-dose AR is superior to the BYHW groups with medium- and low-dose AR groups in inhibiting oxidative stress.