OBJECTIVE: To investigate the effects of astragaloside IV (AS-IV) on podocyte injury of diabetic nephropathy (DN) and reveal its potential mechanism. METHODS: In in vitro experiment, podocytes were divided into 4 groups, normal, high glucose (HG), inositol-requiring enzyme 1 (IRE-1) α activator (HG+thapsigargin 1 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups. Additionally, podocytes were divided into 4 groups, including normal, HG, AS-IV (HG+AS-IV 20 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups, respectively. After 24 h treatment, the morphology of podocytes and endoplasmic reticulum (ER) was observed by electron microscopy. The expressions of glucose-regulated protein 78 (GRP78) and IRE-1α were detected by cellular immunofluorescence. In in vivo experiment, DN rat model was established via a consecutive 3-day intraperitoneal streptozotocin (STZ) injections. A total of 40 rats were assigned into the normal, DN, AS-IV [AS-IV 40 mg/(kg·d)], and IRE-1α inhibitor [STF-083010, 10 mg/(kg·d)] groups (n=10), respectively. The general condition, 24-h urine volume, random blood glucose, urinary protein excretion rate (UAER), urea nitrogen (BUN), and serum creatinine (SCr) levels of rats were measured after 8 weeks of intervention. Pathological changes in the renal tissue were observed by hematoxylin and eosin (HE) staining. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expressions of GRP78, IRE-1α, nuclear factor kappa Bp65 (NF-κBp65), interleukin (IL)-1β, NLR family pyrin domain containing 3 (NLRP3), caspase-1, gasdermin D-N (GSDMD-N), and nephrin at the mRNA and protein levels in vivo and in vitro, respectively. RESULTS: Cytoplasmic vacuolation and ER swelling were observed in the HG and IRE-1α activator groups. Podocyte morphology and ER expansion were improved in AS-IV and IRE-1α inhibitor groups compared with HG group. Cellular immunofluorescence showed that compared with the normal group, the fluorescence intensity of GRP78 and IRE-1α in the HG and IRE-1α activator groups were significantly increased whereas decreased in AS-IV and IRE-1α inhibitor groups (P<0.05). Compared with the normal group, the mRNA and protein expressions of GRP78, IRE-1α, NF-κ Bp65, IL-1β, NLRP3, caspase-1 and GSDMD-N in the HG group was increased (P<0.05). Compared with HG group, the expression of above indices was decreased in the AS-IV and IRE-1α inhibitor groups, and the expression in the IRE-1α activator group was increased (P<0.05). The expression of nephrin was decreased in the HG group, and increased in AS-IV and IRE-1α inhibitor groups (P<0.05). The in vivo experiment results revealed that compared to the normal group, the levels of blood glucose, triglyceride, total cholesterol, BUN, blood creatinine and urinary protein in the DN group were higher (P<0.05). Compared with DN group, the above indices in AS-IV and IRE-1α inhibitor groups were decreased (P<0.05). HE staining revealed glomerular hypertrophy, mesangial widening and mesangial cell proliferation in the renal tissue of the DN group. Compared with the DN group, the above pathological changes in renal tissue of AS-IV and IRE-1α inhibitor groups were alleviated. Quantitative RT-PCR and Western blot results of GRP78, IRE-1α, NF-κ Bp65, IL-1β, NLRP3, caspase-1 and GSDMD-N were consistent with immunofluorescence analysis. CONCLUSION AS-IV could reduce ERS and inflammation, improve podocyte pyroptosis, thus exerting a podocyte-protective effect in DN, through regulating IRE-1α/NF-κ B/NLRP3 signaling pathway.
Podocytes/metabolism* ; Animals ; Diabetic Nephropathies/metabolism* ; Saponins/therapeutic use* ; Triterpenes/therapeutic use* ; Signal Transduction/drug effects* ; NF-kappa B/metabolism* ; Protein Serine-Threonine Kinases/metabolism* ; Male ; Rats, Sprague-Dawley ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Endoribonucleases/metabolism* ; Endoplasmic Reticulum Chaperone BiP ; Rats ; Diabetes Mellitus, Experimental/complications* ; Endoplasmic Reticulum/metabolism* ; Multienzyme Complexes

OBJECTIVE: To investigate the mechanism of electroacupuncture (EA) in treating diabetic gastroparesis (DGP) by inhibiting the activation of Nod-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome and pyroptosis mediated via NLRP3/cysteinyl aspartate specific proteinase-1 (caspase-1)/gasdermin D (GSDMD) signaling pathway. METHODS: Forty Sprague-Dawley rats were randomly divided into 4 groups including the control, DGP model, EA, and MCC950 groups. The DGP model was established by a one-time high-dose intraperitoneal injection of 2% streptozotocin and a high-glucose and high-fat diet for 8 weeks. EA intervention was conducted at Zusanli (ST 36), Liangmen (ST 21) and Sanyinjiao (SP 6) with sparse-dense wave for 15 min, and was administered for 3 courses of 5 days. After intervention, the blood glucose, urine glucose, gastric emptying, and intestinal propulsive rate were observed. Besides, HE staining was used to observe histopathological changes in gastric antrum tissues, and TUNEL staining was utilized to detect DNA damage. Protein expression levels of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), pro-caspase-1, caspase-1 and GSDMD were measured by Western blot. Immunofluorescence staining was employed to assess the activity of GSDMD-N. Lactate dehydrogenase (LDH) levels were detected by using a biochemical kit. RESULTS: DGP rats showed persistent hyperglycemia and a significant decrease in gastrointestinal motility (P<0.05 or P<0.01), accompanied by pathological damage in their gastric antrum tissues. Cellular DNA was obviously damaged, and the expressions of NLRP3, ASC, pro-caspase-1, caspase-1 and GSDMD proteins were significantly elevated, along with enhanced fluorescence signals of GSDMD-N and increased LDH release (P<0.01). EA mitigated hyperglycemia, improved gastrointestinal motility in DGP rats and alleviated their pathological injury (P<0.05). Furthermore, EA reduced cellular DNA damage, lowered the protein levels of NLRP3, ASC, pro-caspase-1, caspase-1 and GSDMD, suppressed GSDMD-N activity, and decreased LDH release (P<0.05 or P<0.01), demonstrating effects comparable to MCC950. CONCLUSION EA promotes gastrointestinal motility and repairs the pathological damage in DGP rats, and its mechanism may be related to the inhibition of NLRP3 inflammasome and pyroptosis mediated by NLRP3/caspase-1/GSDMD pathway.
Animals ; Electroacupuncture ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Pyroptosis ; Rats, Sprague-Dawley ; Caspase 1/metabolism* ; Gastroparesis/physiopathology* ; Signal Transduction ; Male ; Diabetes Mellitus, Experimental/physiopathology* ; Phosphate-Binding Proteins/metabolism* ; Gastrointestinal Motility ; Rats ; Intracellular Signaling Peptides and Proteins/metabolism* ; Diabetes Complications/physiopathology* ; Gasdermins

Diabetic chronic wounds are characterized by difficult healing, recurrent progression, and high rates of disability and mortality, which make their clinical treatment a medical challenge urgent to be addressed. However, the complex local microenvironment conditions of chronic wounds, such as high protease activity and persistent inflammatory responses, result in low bioavailability of exogenous cytokines (e.g., chemokine CXCL12) at the wound site, limiting their clinical application. In this study, we utilized Lactobacillus plantarum WCFS1 as the chassis to develop an efficient CXCL12 delivery system based on synthetic biology. Subsequently, we evaluated the role of the engineered probiotic strain in promoting the chronic wound healing in diabetic mice. Firstly, we fused the endogenous secretion signal peptide lp_3050 (SP_{lp_3050}) of L. plantarum WCFS1 and the commonly used secretion signal peptide usp45 (SP_usp45) of lactic acid bacteria with the reporter gene gusA and inserted them into the pTRK892-P_32(pgm) plasmid by molecular cloning. Then, we prepared the engineered strains and characterized the efficacy of the two signal peptides in driving the secretion of GusA. The results showed that SP_{lp_3050} efficiently drove the secretion of GusA in L. plantarum WCFS1, increasing the activity of GusA in the culture supernatant by nearly five times compared with that of SP_{lp_3050}. Further, we fused SP_{lp_3050} and codon-optimized CXCL12 gene to construct an engineered probiotic strain Lpw-CXCL12 for CXCL12 delivery. The results demonstrated that the content of CXCL12 in the culture supernatant reached (13.40±0.20) μg/mL. Finally, we found that the engineered probiotic strain Lpw-CXCL12 accelerated chronic wound healing in a diabetic mouse model. In conclusion, these results support an engineered probiotic strain in promoting diabetic chronic wound healing, providing a new strategy and technological foundation for the management of diabetic chronic wounds in the future.
Probiotics ; Animals ; Chemokine CXCL12/biosynthesis* ; Mice ; Wound Healing ; Lactobacillus plantarum/metabolism* ; Diabetes Mellitus, Experimental/complications* ; Male

OBJECTIVE: To explore the effects of Qiwei No.3 combined with sildenafil on Rho kinase activity and AKT/eNOS pathways in the penile cavernous tissue of male rats with diabetic erectile dysfunction (DED). METHODS: We constructed a model of DED in 24 SD male rats by intraperitoneal injection of streptozotocin solution and injecting apomorphine into the neck after 8 weeks of feeding, equally randomized the model rats into a model control (MC), a sildenafil (S), a low-dose Qiwei No.3 combined with sildenafil (LQ+S) and a high-dose Qiwei No.3 combined with sildenafil (HQ+S) group, and took another 6 normal male rats as blank controls (BC). We treated intragastrically the animals in the BC and MC groups with normal saline, and those in the S, LQ+S and HQ+S groups with sildenafil (5 mg/kg/d), Qiwei No.3 (10 g/kg/d) + sildenafil (5mg/kg/d), and Qiwei No.3 (20g/kg/d) + sildenafil (5mg/kg/d), respectively. After 6 weeks of treatment, we recorded the number of penile erections of all the rats by injecting apomorphine into the neck, and measured the activity of Rho kinase and expressions of p-AKT and eNOS proteins in the corpus cavernosum by Western blot. RESULTS: Compared with the blank controls, all the DED model rats showed evidently elevated blood glucose and reduced body weight. The number of penile erections was significantly increased in the S, LQ+S and HQ+S groups in comparison with that in the model controls (P< 0.05), even higher in the HQ+S than in the S group (P< 0.05). The activity of Rho kinase in the penile cavernosum was significantly higher in the MC than in the BC group (P<0.05), but lower in the HQ+S than in the S group (P< 0.05). No statistically significant difference was observed in the expression level of the p-AKT protein in the penile cavernosum among the five groups of rats (P > 0.05). The expression of eNOS was remarkably up-regulated in the BC and HQ+S groups (P< 0.05) compared with that in the MC group, even more significantly in the HQ+S than in the LQ+S and S groups (P< 0.05). CONCLUSION The combination of high-dose "Qiwei No. 3" and sildenafil can improve erectile function in DED rats, which may be attributed to its effect of releasing more nitric oxide (NO) by inhibiting the activity of Rho kinase and up-regulating the expression of the e-NOS protein.
Animals ; Male ; Sildenafil Citrate ; Rats ; rho-Associated Kinases/antagonists & inhibitors* ; Rats, Sprague-Dawley ; Penis/drug effects* ; Erectile Dysfunction/etiology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Nitric Oxide Synthase Type III/metabolism* ; Diabetes Mellitus, Experimental/complications* ; Drugs, Chinese Herbal/therapeutic use*

Objective To investigate the effects of simvastatin on diabetic neuropathic pain and systematic inflammation in diabetic rats and explore their molecular mechanisms.Methods Totally 24 rats were equally randomized into the normal+vehicle(NV)group,diabetic+vehicle(DV)group,and diabetic+simvastatin(DS)group using the random number table.Streptozotocin(STZ)was used to establish the rat models of diabetes.Blood glucose,body mass,paw withdrawal mechanical threshold(PWMT),and paw withdrawal thermal latency(PWTL)in each group were observed on days 7,14,21,and 28 after STZ injection.On day 28 after STZ injection,rats were sacrificed,and the lumbar spinal dorsal horn and serum were collected.Western blotting was used to detect the expression of receptor for advanced glycation end products(RAGE)and the phosphorylation levels of protein kinase B(AKT),extracellular signal-regulated kinase(ERK),p38,and c-Jun N-terminal kinase(JNK)in the spinal dorsal horn of rats in each group.Enzyme-linked immunosorbent assay was performed to determine the serum concentrations of oxidized low density lipoprotein(ox-LDL)and interleukin-1β(IL-1β).Results On days 14,21 and 28 after STZ injection,the PWMT in DV group were(8.6 ± 0.8),(7.1 ± 1.6),and(7.8 ± 0.8)g respectively,which were significantly lower than (12.0 ± 0.9)(=8.482, =0.000),(11.6 ± 1.5)(=11.309, =0.000),and(11.7 ± 1.5)g(=9.801, =0.000)in NV group.The PWMT in DS group on days 21 and 28 were(9.4 ± 1.4)(=5.780, =0.000)and(9.7 ± 0.9)g(=4.775, =0.003),respectively,which were significantly improved comparing with those of DV group.On days 7,14,21,and 28,there were no significant differences in PWTL among these three groups (all <0.05).The expression of RAGE in the spinal dorsal horn of DV group was significantly higher than those of NV group(=6.299, =0.000)and DS group(=2.891, =0.025).The phosphorylation level of AKT in the spinal dorsal horn of DV group was significantly higher than those of NV group(=8.915,=0.000)and DS group(=4.103,=0.003).The phosphorylation levels of ERK( =8.313,=0.000),p38( =2.965, =0.022),and JNK(=7.459, =0.000)in the spinal dorsal horn of DV group were significantly higher than those of NV group;the phosphorylation level of JNK in the spinal dorsal horn of DS group was significant lower than that of DV group(=3.866, =0.004);however,there were no significant differences in the phosphorylation levels of ERK(=1.987,=0.122)and p38(=1.260,=0.375)in the spinal dorsal horn between DS group and DV group.The serum concentrations of ox-LDL and IL-1β in DV group were(41.86 ± 13.40)ng/ml and(108.16 ± 25.88)pg/ml,respectively,which were significantly higher than those in NV group [(24.66 ± 7.87)ng/ml(=3.606,=0.003)and(49.32 ± 28.35)pg/ml(=5.079,=0.000)] and DS group [(18.81 ± 5.62)ng/ml (=4.833, =0.000)and(32.73 ± 11.73)pg/ml(=6.510, =0.000)].Conclusions Simvastatin can relieve the mechanical allodynia of diabetic rats possibly by inhibiting the activation of RAGE/AKT and the phosphorylation of JNK in the spinal dorsal horn.Simvastatin can also decrease the serum concentrations of ox-LDL and IL-1β in diabetic rats,which may contribute to the relief of systematic inflammation.
Animals ; Diabetes Mellitus, Experimental ; complications ; Hyperalgesia ; Inflammation ; drug therapy ; Interleukin-1beta ; blood ; Lipoproteins, LDL ; blood ; Neuralgia ; drug therapy ; Proto-Oncogene Proteins c-akt ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptor for Advanced Glycation End Products ; metabolism ; Simvastatin ; pharmacology

Diabetic cardiomyopathy( DCM) is one of the major cardiovascular complications of diabetes mellitus. Based on the clinical efficacy of Danzhi Jiangtang Capsules( DJC) in the prevention and treatment of diabetes and its cardiovascular complications,both in vivo and in vitro methods were adopted to investigate its effect and underlying mechanism of protecting myocardial injury induced by diabetes. The type 2 diabetic rats were prepared by feeding high-energy food combined with streptozotin( STZ) injection,and the effects of DJC were observed by blood sugar,blood lipid,hemodynamic index,cardiac weight index and the change of cardiac pathological morphology. The protein expressions of TLR4,MyD88 and NF-κB p65 in myocardial tissue were detected and the possible mechanism was preliminarily analyzed. Besides this,DJC containing serum was prepared,H9 c2 cardiomyocyte induced by high sugar were studied to investigate the mechanism of TLR4/MyD88/NF-κB signaling pathway regulating cardiomyocyte injury and the therapeutic effect of DJC. The results demonstrated that fasting blood sugar,glycosylated hemoglobin,total cholesterol and glycerol triglyceride were significantly reduced( P<0. 01,P<0. 05). Cardiac weight index,left ventricle weight index,LVEDP and the protein expressions of TLR4,MyD88 and NF-κB p65 were significantly reduced( P<0. 01,P<0. 05). LVSP,+dp/dtmaxand-dp/dtmaxincreased significantly( P<0. 01,P< 0. 05). Moreover,the pathological damage of myocardial tissue in rats improved significantly. Meanwhile,the protein expressions of TLR4,MyD88 and NF-κB p65 in cardiomyocytes induced by high sugar were significantly inhibited( P<0. 01).It showed that DJC were effective in preventing and treating myocardial injury induced by diabetes and its mechanism may be related to the over-expression of TLR4/MyD88/NF-κB signaling pathway induced by high sugar.
Animals ; Blood Glucose ; Capsules ; Diabetes Mellitus, Experimental/complications* ; Diabetic Cardiomyopathies/drug therapy* ; Drugs, Chinese Herbal/therapeutic use* ; Myeloid Differentiation Factor 88/metabolism* ; NF-kappa B/metabolism* ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Toll-Like Receptor 4/metabolism*

Calpain activation contributes to hyperglycemia-induced endothelial dysfunction and apoptosis. This study was designed to investigate the role of calpain inhibition in improving diabetic erectile dysfunction (ED) in mice. Thirty-eight-week-old male C57BL/6J mice were divided into three groups: (1) nondiabetic control group, (2) diabetic mice + vehicle group, and (3) diabetic mice + MDL28170 (an inhibitor of calpain) group. Type 1 diabetes was induced by intraperitoneal injection of streptozotocin at 60 mg kg^-1 body weight for 5 consecutive days. Thirteen weeks later, diabetic mice were treated with MDL28170 or vehicle for 4 weeks. The erectile function was assessed by electrical stimulation of the cavernous nerve. Penile tissues were collected for measurement of calpain activity and the endothelial nitric oxide synthase (eNOS)-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. Terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining was used to evaluate apoptosis. Caspase-3 expression and activity were also measured to determine apoptosis. Our results showed that erectile function was enhanced by MDL28170 treatment in diabetic mice compared with the vehicle diabetic group. No differences in calpain-1 and calpain-2 expressions were observed among the three groups. However, calpain activity was increased in the diabetic group and reduced by MDL28170. The eNOS-NO-cGMP pathway was upregulated by MDL28170 treatment in diabetic mice. Additionally, MDL28170 could attenuate apoptosis and increase the endothelium and smooth muscle levels in corpus cavernosum. Inhibition of calpain could improve erectile function, probably by upregulating the eNOS-NO-cGMP pathway and reducing apoptosis.
Animals ; Apoptosis/drug effects* ; Calpain/antagonists & inhibitors* ; Cyclic GMP/biosynthesis* ; Diabetes Complications/drug therapy* ; Diabetes Mellitus, Experimental/complications* ; Dipeptides/therapeutic use* ; Endothelium/metabolism* ; Enzyme Inhibitors/therapeutic use* ; Erectile Dysfunction/etiology* ; In Situ Nick-End Labeling ; Male ; Mice ; Mice, Inbred C57BL ; Muscle, Smooth/metabolism* ; Nitric Oxide Synthase Type III/biosynthesis* ; Penis/enzymology* ; Up-Regulation

The study aimed to investigate the intervening role of Didang decoction (DDD) at different times in macrovascular endothelial defense function, focusing on its effects on the AMP-activated protein kinase (AMPK) signaling pathway. The effects of DDD on mitochondrial energy metabolism were also investigated in rat aortic endothelial cells (RAECs). Type 2 diabetes were induced in rats by streptozotocin (STZ) combined with high fat diet. Rats were randomly divided into non-intervention group, metformin group, simvastatin group, and early-, middle-, late-stage DDD groups. Normal rats were used as control. All the rats received 12 weeks of intervention or control treatment. Western blots were used to detect the expression of AMP-activated protein kinase α1 (AMPKα1) and peroxisome proliferator-activated receptor 1α (PGC-1α). Changes in the intracellular AMP and ATP levels were detected with ELISA. Real-time-PCR was used to detect the mRNA level of caspase-3, endothelial nitric oxide synthase (eNOS), and Bcl-2. Compared to the diabetic non-intervention group, a significant increase in the expression of AMPKα1 and PGC-1α were observed in the early-stage, middle-stage DDD groups and simvastatin group (P < 0.05). The levels of Bcl-2, eNOS, and ATP were significantly increased (P < 0.05), while the level of AMP and caspase-3 were decreased (P < 0.05) in the early-stage DDD group and simvastatin group. Early intervention with DDD enhances mitochondrial energy metabolism by regulating the AMPK signaling pathway and therefore may play a role in strengthening the defense function of large vascular endothelial cells and postpone the development of macrovascular diseases in diabetes.
AMP-Activated Protein Kinases ; metabolism ; Adenosine Triphosphate ; metabolism ; Animals ; Aorta ; drug effects ; metabolism ; Cardiovascular Diseases ; metabolism ; prevention & control ; Caspase 3 ; metabolism ; Diabetes Mellitus, Experimental ; complications ; drug therapy ; metabolism ; Diabetes Mellitus, Type 2 ; complications ; drug therapy ; metabolism ; Diptera ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Endothelial Cells ; drug effects ; metabolism ; Endothelium, Vascular ; drug effects ; metabolism ; Energy Metabolism ; drug effects ; Leeches ; Mitochondria ; drug effects ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; metabolism ; Phytotherapy ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Prunus persica ; Rats, Sprague-Dawley ; Rheum ; Signal Transduction

OBJECTIVETo investigate the effect of Xuezhikang (, XZK) on renal cell apoptosis in diabetic rats and the possible mechanism.

METHODSSixty-six rats were randomly divided into 3 groups: the normal, model and XZK groups. In each group, the rats were further randomly divided into 3-month and 6-month subgroups, respectively. Diabetes of rats was induced by a single intraperitoneal injection of 1% streptozocin at 60 mg/kg body weight. Rats in the XZK group received gastric perfusion of XZK (1200 mg/kg body weight) everyday for 3 or 6 months, while rats in the normal and model groups received equal volume of saline. Twenty-four hours' urine was collected for urinary albumin excretion (UAE) measurement. Periodic acid-Schiff (PAS) and Masson's trichrome staining were used for saccharides and collagen detection. Cell apoptosis of renal cortex was investigated by TdT-mediated dUTP nick end labeling (TUNEL) staining. Bax and Bcl-2 expressions were detected by immunohistochemistry and Western blot, respectively. Cytochrome C (Cyt C) and caspase-9 concentration were detected by Western blot.

RESULTSCompared with the model group, XZK treatment could significantly decrease the kidney hypertrophy index, 24 h UAE, renal cell apoptosis, cytoplasmic Cyt C level and active caspase-9 level, as well as suppress the increment of Bax and up-regulate the expression of Bcl-2, leading to the suppression of Bax/Bcl-2 ratio at 3 and 6 months (P<0.05 or P<0.01). Moreover, XZK treatment could alleviate the deposition of PAS-stained saccharides and Masson's trichromestained collagen to different extent.

CONCLUSIONSRenal cell apoptosis was observed in diabetic kidney, in which mitochondrial apoptotic pathway might be involved. XZK treatment could attenuate pathological changes in diabetic kidney and reduce renal cell apoptosis, probably via the suppression of Bax/Bcl-2 ratio, which lead to inhibition of Cyt C release and following caspase-9 activation.

Albuminuria ; blood ; complications ; Animals ; Apoptosis ; drug effects ; Blood Glucose ; metabolism ; Caspase 9 ; metabolism ; Cytochromes c ; metabolism ; Diabetes Mellitus, Experimental ; blood ; drug therapy ; metabolism ; pathology ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Hypertrophy ; In Situ Nick-End Labeling ; Kidney ; drug effects ; pathology ; Kidney Glomerulus ; pathology ; Lipids ; blood ; Male ; Mesangial Cells ; drug effects ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats, Sprague-Dawley ; Streptozocin ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo observe the deregulation of autophagy in diabetic peripheral neuropathy (DPN) and investigate whether Jinmaitong ( JMT) alleviates DPN by inducing autophagy.

METHODSDPN models were established by streptozotocin-induced diabetic rats and Schwann cells (SCs) cultured in high glucose medium. The pathological morphology was observed by the improved Bielschowsky's nerve fiber axonal staining and the Luxol fast blue-neutral red myelin staining. The ultrastructure was observed by the transmission electron microscopy. Beclin1 level was detected by immunohistochemistry and Western blot. The proliferation of cultured SCs was detected by methylthiazolyldiphenyl-tetrazolium bromide.

RESULTSDiabetic peripheral nerve tissues demonstrated pathological morphology and reduced autophagic structure, accompanied with down-regulation of Beclin1. JMT apparently alleviated the pathological morphology change and increased the autophagy [in vivo, Beclin1 integral optical density (IOD) value of the control group 86.6±17.7, DM 43.9±8.8, JMT 73.3 ±17.8, P<0.01 or P<0.05, in vitro Beclin1 IOD value of the glucose group 0.47±0.25 vs the control group 0.88±0.29, P<0.05]. Consequently, inhibition of autophagy by 3-methyladenine resulted in a time- and concentration-dependent decrease of the proliferation of SCs (P<0.05, P<0.01).

CONCLUSIONSDown-regulation of autophagy in SCs might contribute to the pathogenesis of DPN. JMT alleviates diabetic peripheral nerve injury at least in part by inducing autophagy.

Animals ; Autophagy ; drug effects ; Axons ; drug effects ; pathology ; Beclin-1 ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Diabetes Mellitus, Experimental ; complications ; drug therapy ; pathology ; Diabetic Neuropathies ; complications ; drug therapy ; pathology ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Glucose ; pharmacology ; Immunohistochemistry ; Male ; Rats, Wistar ; Schwann Cells ; drug effects ; pathology ; Sciatic Nerve ; drug effects ; pathology ; ultrastructure ; Staining and Labeling