OBJECTIVES: To investigate the effects and molecular mechanisms of asiatic acid on β-cell function in type 2 diabetes mellitus (T2DM). METHODS: The T2DM model was established by high fat diet and streptozotocin injection in ICR mice, and the effects of asiatic acid on glucose regulation were investigated in model mice. The islets were isolated from palmitic acid-treated diabetic mice. ELISA was used to detect the glucose-stimulated insulin secretion, tumor necrosis factor (TNF)-α and interleukin (IL)-6. ATP assay was applied to measure ATP production, and Western blotting was used to detect protein expression of mature β cell marker urocortin (Ucn) 3 and mitofusin (Mfn) 2. The regulatory effects of asiatic acid on glucose-stimulated insulin secretion (GSIS) and Ucn3 expression were also investigated after siRNA interference with Mfn2 or treatment with TNF-α. RESULTS: Asiatic acid with the dose of 25 mg·kg^－1·d^－1 had the best glycemic control in T2DM mice and improved the homeostasis model assessment β index. Asiatic acid increased the expression of Mfn2 and Ucn3 protein and improved the GSIS function of diabetic β cells in vitro and in vivo (both P<0.05). Moreover, it improved the ATP production of islets of T2DM mice in vitro (P<0.05). Interfering Mfn2 with siRNA blocked the up-regulation of Ucn3 and GSIS induced by asiatic acid. Asiatic acid inhibited islet TNF-α content and increased Mfn2 and Ucn3 protein expression inhibited by TNF-α. CONCLUSIONS Asiatic acid improves β cell insulin secretion function in T2DM mice by maintaining the β cell maturity, which may be related to the TNF-α/Mfn2 pathway.
Mice ; Animals ; Insulin Secretion ; Diabetes Mellitus, Type 2/drug therapy* ; Islets of Langerhans/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Insulin/therapeutic use* ; Diabetes Mellitus, Experimental ; Mice, Inbred ICR ; Glucose/therapeutic use* ; Interleukin-6/metabolism* ; RNA, Small Interfering/pharmacology* ; Adenosine Triphosphate ; GTP Phosphohydrolases/therapeutic use*

Tu-Xian decoction (TXD), a traditional Chinese medicine (TCM) formula, has been frequently administered to manage diabetic cognitive impairment (DCI). Despite its widespread use, the mechanisms underlying TXD's protective effects on DCI have yet to be fully elucidated. As a significant regulator in neurodegenerative conditions, death-associated protein kinase-1 (DAPK-1) serves as a focus for understanding the action of TXD. This study was designed to whether TXD mediates its beneficial outcomes by inhibiting DAPK-1. To this end, a diabetic model was established using Sprague-Dawley (SD) rats through a high-fat, high-sugar (HFHS) diet regimen, followed by streptozotocin (STZ) injection. The experimental cohort was stratified into six groups: Control, Diabetic, TC-DAPK6, high-dose TXD, medium-dose TXD, and low-dose TXD groups. Following a 12-week treatment period, various assessments-including blood glucose levels, body weight measurements, Morris water maze (MWM) testing for cognitive function, brain magnetic resonance imaging (MRI), and histological analyses using hematoxylin-eosin (H&E), and Nissl staining-were conducted. Protein expression in the hippocampus was quantified through Western blotting analysis. The results revealed that TXD significantly improved spatial learning and memory abilities, and preserved hippocampal structure in diabetic rats. Importantly, TXD administration led to a down-regulation of proteins indicative of neurological damage and suppressed DAPK-1 activity within the hippocampal region. These results underscore TXD's potential in mitigating DCIvia DAPK-1 inhibition, positioning it as a viable therapeutic candidate for addressing this condition. Further investigation into TXD's molecular mechanisms may elucidate new pathways for the treatment of DCI.
Animals ; Rats ; Brain/metabolism* ; Cognitive Dysfunction/drug therapy* ; Diabetes Mellitus, Experimental/metabolism* ; Hippocampus ; Rats, Sprague-Dawley

This study investigated the effect of Lianmei Qiwu Decoction(LMQWD) on the improvement of cardiac autonomic nerve remodeling in the diabetic rat model induced by the high-fat diet and explored the underlying mechanism of LMQWD through the AMP-activated protein kinase(AMPK)/tropomyosin receptor kinase A(TrkA)/transient receptor potential melastatin 7(TRPM7) signaling pathway. The diabetic rats were randomly divided into a model group, an LMQWD group, an AMPK agonist group, an unloaded TRPM7 adenovirus group(TRPM7-N), an overexpressed TRPM7 adenovirus group(TRPM7), an LMQWD + unloaded TRPM7 adenovirus group(LMQWD+TRPM7-N), an LMQWD + overexpressed TRPM7 adenovirus group(LMQWD+TRPM7), and a TRPM7 channel inhibitor group(TRPM7 inhibitor). After four weeks of treatment, programmed electrical stimulation(PES) was employed to detect the arrhythmia susceptibility of rats. The myocardial cell structure and myocardial tissue fibrosis of myocardial and ganglion samples in diabetic rats were observed by hematoxylin-eosin(HE) staining and Masson staining. The immunohistochemistry, immunofluorescence, real-time quantitative polymerase chain reaction(RT-PCR), and Western blot were adopted to detect the distribution and expression of TRPM7, tyrosine hydroxylase(TH), choline acetyltransferase(ChAT), growth associated protein-43(GAP-43), nerve growth factor(NGF), p-AMPK/AMPK, and other genes and related neural markers. The results showed that LMQWD could significantly reduce the arrhythmia susceptibility and the degree of fibrosis in myocardial tissues, decrease the levels of TH, ChAT, and GAP-43 in the myocardium and ganglion, increase NGF, inhibit the expression of TRPM7, and up-regulate p-AMPK/AMPK and p-TrkA/TrkA levels. This study indicated that LMQWD could attenuate cardiac autonomic nerve remodeling in the diabetic state, and its mechanism was associated with the activation of AMPK, further phosphorylation of TrkA, and inhibition of TRPM7 expression.
Rats ; Animals ; AMP-Activated Protein Kinases/metabolism* ; Nerve Growth Factor/metabolism* ; Diabetes Mellitus, Experimental/drug therapy* ; TRPM Cation Channels/metabolism* ; GAP-43 Protein/metabolism* ; Signal Transduction ; Diabetic Neuropathies/genetics* ; Fibrosis

Objective To investigate the neuroprotective effect of methylene blue on diabetic retinopathy in rats. Methods Thirty SD rats were randomly divided into blank, control and experimental groups. The control and experimental groups were induced with diabetes by streptozotocin (STZ) intraperitoneal injection. After 6 weeks of successful modeling, the experimental group received intravitreal injection of methylene blue at a dose of [0.2 mg/(kg.d)], while the control group received an equal amount of dimethyl sulfoxide (DMSO) intravitreal injection, both continuously injected for 7 days. ELISA was used to detect the levels of retinal superoxide dismutase (SOD), 8-iso-prostaglandin F2alpha (iPF2α) and interleukin-1β (IL-1β) in rats. Western blot analysis was used to detect the expression of retinal extracellular signal-regulated kinase 1/2 phosphorylation (p-ERK1/2) and phosphorylated protein kinase B (p-AKT), and PAS staining was used to detect retinal morphological changes. Results Compared with the blank group rats, the retinal SOD activity in the control and experimental group rats was significantly reduced. iPF2α, IL-1β and p-ERK1/2 level increased, while p-AKT level decreased. Compared with the control group, the SOD activity of the experimental group rats increased. iPF2α and IL-1β level went down, while p-ERK1/2 and p-AKT level went up significantly. The overall thickness of the retinal layer and the number of retinal ganglion cells were significantly reduced. Conclusion Methylene blue improves diabetic retinopathy in rats by reducing retinal oxidative stress and enhancing ERK1/2 and AKT phosphorylation.
Rats ; Animals ; Diabetic Retinopathy/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Mitogen-Activated Protein Kinase 3/metabolism* ; Interleukin-1beta/metabolism* ; Methylene Blue/pharmacology* ; Phosphorylation ; Rats, Sprague-Dawley ; MAP Kinase Signaling System ; Diabetes Mellitus, Experimental/drug therapy* ; Superoxide Dismutase/metabolism*

Diabetic nephropathy (DN) is one of the most common complications of diabetes mellitus, which is characterized in renal tubulointerstitial fibrosis (TIF). The current study was designed to investigate the protective effect of Jujuboside A (Ju A) on TIF in type 2 diabetes (T2DM) mice, and explore its underlying anti-fibrosis mechanism. A mouse T2DM model was established using high fat diet (HFD) feeding combined with intraperitoneal injection of streptozotocin (STZ). Then, diabetic mice were treated with Ju A (10, 20 and 40 mg·kg^-1·d^-1, i.g.) for 12 weeks. Results showed that administration of Ju A not only down-regulated fasting blood glucose (FBG) levels, but also improved hyperlipidemia and renal function in diabetic mice. Moreover, the reduced ECM accumulation was observed in the renal cortex of Ju A treated diabetic mice, while the TIF progression was also attenuated by Ju A through blocking the epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells (RTECs). Further mechanism studies showed that Ju A treatment effectively down-regulated the protein expression and subsequent nuclear translocation of Yin Yang 1 (YY1) in the renal cortex of diabetic mice, and reduced the levels of transforming growth factor-β1 (TGF-β1) in the serum and renal cortex of Ju A treated mice. According to invitro studies, the up-regulated YY1/TGF-β1 signaling pathway was restored by Ju A in high glucose (HG) cultured HK-2 cells. Taken together, these findings demonstrated that Ju A can ameliorate the TIF of DN through down-regulating the YY1/TGF-β1 signaling pathway.
Animals ; Blood Glucose ; Diabetes Mellitus, Experimental/metabolism* ; Diabetes Mellitus, Type 2/drug therapy* ; Diabetic Nephropathies/metabolism* ; Fibrosis ; Mice ; Saponins ; Signal Transduction ; Streptozocin ; Transforming Growth Factor beta1/metabolism*

OBJECTIVE: To investigate the protective effects of modified Linggui Zhugan Decoction (, MLZD), a traditional Chinese medicine formula, on obese type 2 diabetes mellitus (T2DM) rats. METHODS: Fifty Sprague-Dawley rats were randomly divided into 5 groups by a random number table, including normal, obese T2DM (ob-T2DM), MLZD low-dose [MLDZ-L, 4.625 g/(kg·d)], MLZD middle-dose [MLD-M, 9.25 g/(kg·d) ] and MLZD high-dose [MLD-H, 18.5 g/(kg·d)] groups, 10 rats in each group. After 4-week intervention, blood samples and liver, pancreas, muscle tissues were collected to assess the insulin resistance (IR), blood lipid, adipokines and inflammation cytokines. The alteration of phosphatidylinositol 3 kinase (PI3K)-protein kinase B (PKB or Akt)/the mammalian target of rapamycin (mTOR)-ribosome protein subunit 6 kinase 1 (S6K1 )/AMP-activated protein kinase (AMPK)-peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 α) pathways were also studied. RESULTS: MLZD dose-dependently reduced fasting blood glucose, fasting insulin, homeostasis model of assessment for IR index and increased insulin sensitive index compared with ob-T2DM rats (P<0.05). Similarly, total cholesterol, triglyceride, low-density lipoprotein cholesterol and free fatty acids were also decreased compared with ob-T2DM rats after 4-week treatment (P<0.05 or P<0.01). Improvements in adipokines and inflammatory cytokines were observed with a raised level of adiponectin and a reduced level of leptin, resistin, tumor necrosis factor-α and interleukin-6 (P<0.05 or P<0.01). MLZD regulated the PI3K-Akt/mTOR-S6K1/AMPK-PGC-1 α pathways and restored the tissue structure of liver and pancreas (P<0.05 or P<0.01). CONCLUSIONS MLZD ameliorated glycolipid metabolism and inflammation, which may be attributed to the regulation of PI3K-Akt/mTOR-S6K1/AMPK-PGC-1 α pathways.
AMP-Activated Protein Kinases/metabolism* ; Animals ; Diabetes Mellitus, Experimental ; Diabetes Mellitus, Type 2/drug therapy* ; Glycolipids ; Inflammation ; Obesity/drug therapy* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism*

The dry root and rhizome of Panax ginseng C. A. Mey has garnered much interest owing to its medicinal properties against diabetes and cardiovascular diseases. In this study, an ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS)-based metabolomics approach was used to illustrate the therapeutic mechanisms of ginseng extract on the serum and urinary metabolic profiles in streptozotocin-induced type 1 diabetes mellitus (T1DM) rats. Pharmacological and renal parameters in response to the administration of ginseng were also evaluated. In total, 16 serum endogenous metabolites and 14 urine endogenous metabolites, including pyruvic acid, indoleacetic acid, and phenylacetylglycine, were identified as potential biomarkers for diabetes. Pathway enrichment and network analysis revealed that the biomarkers modulated by ginseng were primarily involved in phenylalanine and pyruvate metabolism, as well as in arginine biosynthesis. Moreover, the levels of several renal injury-related biomarkers in T1DM rats were significantly restored following treatment with ginseng. The administration of the extract helped maintain tissue structure integrity and ameliorated renal injury. The findings suggest that the regulatory effect of ginseng extract on T1DM involves metabolic management of diabetic rats, which subsequently attenuates T1DM-induced early renal dysfunction.
Animals ; Biomarkers ; Chromatography, High Pressure Liquid/methods* ; Diabetes Mellitus, Experimental/metabolism* ; Diabetes Mellitus, Type 1/drug therapy* ; Kidney ; Metabolomics/methods* ; Panax/chemistry* ; Plant Extracts/pharmacology* ; Rats

This study focused on the ameliorative effects of gypenosides(GPS) on insulin sensitivity and inflammatory factors in rats with type 2 diabetes mellitus(T2 DM) and explored their possible molecular mechanisms. After the successful establishment of T2 DM model, diabetic rats were randomly divided into four groups, including model group, GPS groups(200, 100 mg·kg~(-1)) and metformin group(100 mg·kg~(-1)), with healthy rats serving as the control. After 6-week intragastric administration, fasting blood glucose(FBG) and oral glucose tolerance were examined. The levels of insulin, C-peptide, tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6(IL-6) and C-reactive protein(CRP) in serum were examined. Then the homeostasis model assessment of insulin resistance(HOMA-IR) and insulin sensitivity index(ISI) were calculated. The protein expression levels of phosphorylated insulin receptor substrate-1(p-IRS-1) and phosphorylated protein kinase B(p-Akt) in skeletal muscle were measured by Western blot, as well as those of phosphorylated inhibitor of nuclear factor-κB(NF-κB) kinase β(p-IKKβ), phosphorylated alpha inhibitor of NF-κB(p-IκBα) and phosphorylated p65 subunit of NF-κB(p-p65) in adipose tissue. The relative expression levels of glucose transporter 4(GLUT4) mRNA in skeletal muscle and NF-κB mRNA in adipose tissue were measured by qRT-PCR, and the morphological changes of pancreatic tissue were observed. Compared with the model group, the GPS groups witnessed significant decrease in FBG, marked amelioration of impaired oral glucose tolerance and significant increase in ISI. Further, the high-dose GPS group saw significantly reduced HOMA-IR, TNF-α, IL-1β and CRP, significantly increased expression levels of p-IRS-1(Tyr), p-Akt and GLUT4, and markedly inhibited p-IRS-1(Ser), p-IKKβ, p-IκBα, p-p65 and NF-κB. The concentration of CRP and the expression levels of p-IRS-1(Ser), p-IKKβ, p-IκBα and NF-κB were remarkably reduced in the low-dose GPS group. However, GPS was found less effective in the regulation of serum insulin, C-peptide and IL-6 levels and the alleviation of pancreatic islet injury. The results indicated that GPS can reduce FBG and improve insulin sensitivity in diabetic rats possibly by regulating the NF-κB signaling pathway, inhibiting inflammation, and thereby regulating the expression of key proteins in the insulin signaling pathway.
Animals ; Diabetes Mellitus, Experimental/drug therapy* ; Diabetes Mellitus, Type 2/genetics* ; Gynostemma ; Insulin ; Insulin Resistance ; NF-kappa B/metabolism* ; Plant Extracts ; Rats ; Signal Transduction

This study investigated the differential mechanisms of Rehmanniae Radix and Rehmanniae Radix Praeparata in improving diabetes in mice through AMPK-mediated NF-κB/NLRP3 signaling pathway. The diabetic mouse model was established with high-fat diet coupled with streptozotocin(STZ, intraperitoneal injection, 100 mg·kg~(-1), once a day for three consecutive days), after which the mice were randomly divided into model group, low-dose(5 g·kg~(-1)) and high-dose(15 g·kg~(-1)) Rehmanniae Radix groups, low-dose(5 g·kg~(-1)) and high-dose(15 g·kg~(-1)) Rehmanniae Radix Praeparata groups, catalpol group(250 mg·kg~(-1)), 5-hydroxymethylfurfural(5-HMF) group(250 mg·kg~(-1)), metformin group(250 mg·kg~(-1)), with the normal group also set. The organ indexes of heart,liver, spleen, lung, kidney and pancreas were calculated after four weeks of administration. The pathological changes and fibrosis of pancreas, kidney and liver in mice were observed by hematoxylin-eosin(HE) staining and Masson staining. Western blot was used to determine the expression levels of Toll-like receptor-4(TLR4), nuclear factor-κB(NF-κB), Nod-like receptor protein 3(NLRP3),interleukin-1β(IL-1β), adenosine monophosphate-activated protein kinase(AMPK), phosphorylated AMPK(p-AMPK) in the pancreas, kidney and liver of mice. Compared with the model group, the administration groups witnessed significant decrease in the liver,spleen, kidney, pancreas and fat indexes of diabetic mice, and there was no significant difference in heart and lung indexes. The pathological states and fibrosis of pancreatic, kidney and liver tissues were significantly improved after administration. Additionally, the expression levels of TLR4, NF-κB and NLRP3 in pancreas, kidney and liver of diabetic mice were significantly lowered. The expression levels of p-AMPK/AMPK were enhanced significantly in kidney and liver of mice in Rehmanniae Radix group while in pancreas, kidney and liver in Rehmanniae Radix Praeparata group. This suggests that Rehmanniae Radix and Rehmanniae Radix Praeparata differ in the mechanism of regulating energy metabolism of multiple organs and thereby exerting anti-inflammatory effects to alleviate symptoms of diabetic mice.
AMP-Activated Protein Kinases/genetics* ; Animals ; Diabetes Mellitus, Experimental/drug therapy* ; Diet, High-Fat/adverse effects* ; Mice ; NF-kappa B/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein ; Plant Extracts ; Rehmannia ; Signal Transduction ; Streptozocin

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