Peptides obtained by enzymatic decomposition of mackerel induce recovery from physical fatigue by enhancing the SIRT1-mediated antioxidant effect in the soleus muscle of mice.

Fatigue is a serious health problem, and long-term fatigue can lead to mental illnesses and accelerated aging. Oxidative stress, which causes excessive production of reactive oxygen species, is generally thought to increase during exercise and is an indicator of fatigue. Peptides obtained by enzymatic decomposition of mackerel (EMP) contain selenoneine, a strong antioxidant. Although antioxidants increase endurance, the effects of EMP on physical fatigue are unknown. The present study aimed to clarify this aspect. We investigated the effects of EMP on changes in locomotor activity, expression levels of silent mating type information regulation 2 homolog peroxisome 1 (SIRT1), proliferator-activated receptor-γ coactivator-1α (PGC1α), and antioxidative-related proteins including superoxide dismutase 1 (SOD1), SOD2, glutathione peroxidase 1, and catalase in the soleus muscle following EMP treatment before and/or after forced walking. Treatment with EMP before and after forced walking, and not only at one or another time point, improved the subsequent decrease in the locomotor activity and enhanced the levels of SIRT1, PGC1α, SOD1, and catalase expression in the soleus muscle of mice. Moreover, EX-527, a SIRT1 inhibitor, abolished these effects of EMP. Thus, we suggest that EMP combats fatigue by modulating the SIRT1/PGC1α/SOD1-catalase pathway.

Effect of Globin Digests on Physical Fatigue in Mice.

Globin digest (GD) inhibits dietary hypertriglyceridemia; however, its effects on physical fatigue remain unknown. Therefore, this study aimed to investigate the potential anti-fatigue effects of GD. Repeated administration of GD and valine (Val)-Val-tyrosine (Tyr)-proline (Pro), a component of GD, for five days prevented the forced walking-induced decrease in locomotion. Furthermore, GD treatment reversed the forced walking-induced increase in blood lactate levels in mice and increased phosphorylated AMP-activated protein kinase (p-AMPK) in the soleus muscle, suggesting that the anti-fatigue effect of GD involves AMPK activation in the soleus muscle through reduced blood lactate.

ERK5 inhibitor BIX02189 attenuates methamphetamine-induced hyperactivity by modulating microglial activation in the striatum.

Extracellular signal-regulated protein kinase 5 (ERK5) has various physiological functions. However, the physiological role of ERK5 in the treatment of mice with an illicit drug such as methamphetamine (METH) remains unknown. We revealed that mice treated with METH showed hyperactivity, and increased p-ERK5 and Iba1 (a microglia marker) levels in the striatum. Additionally, these changes were inhibited by pretreatment with the ERK5 inhibitor BIX02189. The results suggest that METH-induced hyperactivity is associated with the activation of microglia via p-ERK5 in the striatum. Thus, the ERK5 pathway components in the central nervous system are potential therapeutic targets for preventing METH addiction.

Low Skeletal Muscle Mass Is Associated With Perioperative Neurocognitive Disorder Due To Decreased Neurogenesis in Rats.

BACKGROUND: Perioperative neurocognitive disorder (PND) is a postsurgical complication associated with neuroinflammation and impaired hippocampal neurogenesis, in which brain-derived neurotrophic factor (BDNF) plays a key role. Sarcopenia refers to age-related muscle loss that causes cognitive decline, muscle atrophy, and postoperative delirium. Rats with tail suspension (TS) were used to represent a low-activity model, which involves decreased hind limb function by TS. This hind limb unloading by TS can induce sarcopenia in 2 weeks. However, the relationship between PND and muscle atrophy is unclear. In this experiment, we investigated whether preoperative muscle atrophy induced by TS would affect neurogenesis and accelerate PND in rats. METHODS: Sixty 21-week-old rats were assigned to 4 groups: the TS group, the TS with surgery (TS + S) group, the control group, and the control with surgery (control + S) group. After the abdominal manipulation under 3% sevoflurane anesthesia, cognitive function was assessed using the Morris water maze test and a fear-conditioning test. Neurogenesis was evaluated by checking BDNF secretion and immunohistochemical staining in the hippocampus. RESULTS: The TS + S group showed impaired swimming latency (difference of means = 12.4 versus control + S; 95% confidence interval [CI], 2.0-22.7; P = .016) (difference of means = 15.2 versus TS; 95% CI, 0.4-30.1; P = .043) and path length (difference of means = 147.8 versus control + S; 95% CI, 20.7-274.9; P = .020) in the maze test and cued fear memory (difference of means = -26.0 versus TS; 95% CI, -46.4 to -5.6; P = .006) (difference of means = -22.3 versus control + S; 95% CI, -42.7 to -1.9; P = .026) in the fear-conditioning test. The postoperative levels of BDNF in the TS + S and TS groups were reduced compared with the other groups (P = .002). The number of neural precursors in the dentate gyrus was significantly lower in the TS + S group (P < .001). CONCLUSIONS: We observed that preoperative hind limb muscle atrophy, indicated by TS, was associated with an increased occurrence of PND through the reduction in BDNF and neurogenesis after abdominal surgery in young adult rats. Therefore, we concluded that preoperative low skeletal muscle mass can induce PND due to impaired postoperative neurogenesis. Our findings might indicate that low-cost perioperative interventions, such as preoperative exercise, is beneficial to preventing PND.

Antidepressant Effect of Intracerebroventricularly Administered Deltorphin Analogs in the Mouse Tail Suspension Test.

Several studies have proposed δ opioid receptors as influential targets for developing novel antidepressants. Deltorphin (DLT) and deltorphin II (DLT-II) have high affinity and selectivity for δ opioid receptors; thus, it is likely that DLT analogs possess antidepressant-like effects. Based on this, we evaluated the effects of four DLT analogs (DLT-related synthetic peptides) on immobility behavior in the tail suspension test. Intracerebroventricular administration of DLT or [N-isobutyl-Gly6]DLT in mice significantly decreased immobile behavior. However, administration of DLT did not affect locomotor activity, whereas that of [N-isobutyl-Gly6]DLT significantly increased locomotion in mice. The effect of the shortened immobility time following DLT administration was counteracted by the administration of the selective δ1 opioid receptor antagonist 7-benzylidenenaltrexone, but not by the selective δ2 opioid receptor antagonist naltriben. These findings suggest that DLT has an antidepressant-like effect by activating the central δ1 opioid receptor in mice.

Intracerebroventricular Administration of Dermorphin-Dynorphin Analogs Producing Antidepressant-Like Effects through Activation of µ1- and κ-Opioid Receptors in Mice.

The opioid system in the central nervous system regulates depressive-like behavior in animals. Opioid receptors and their endogenous ligands have been focused on as novel therapeutic targets for depression. We synthesized dermorphin (DRM)-dynorphin (DYN) analogs (DRM-DYN001-004) using the message-address concept concerning interactions with opioid receptors. It has previously been reported that DRM-DYN001, 003, and 004 have shown high affinities for µ- and κ-opioid receptors, whereas all analogs had a lower affinity for the δ-opioid receptor than for other receptors using a receptor binding assay. However, it remains unknown whether these analogs show antidepressant-like effects in mice. We examined the effects of DRM-DYN analogs on the duration of immobile behavior in a tail suspension test. Intracerebroventricular administration of DRM-DYN001 in mice shortened the duration of immobile behavior, but did not affect locomotion. The DRM-DYN001-induced antidepressant-like effect was inhibited by co-administration of naloxone (non-selective opioid receptor antagonist), naloxonazine (selective µ1-opioid receptor antagonist), or nor-BNI (κ-opioid receptor antagonist), but not naltrindole (δ-opioid receptor antagonist). These data suggest that DRM-DYN001 exerts an antidepressant-like effect via activation of the central µ1- and κ-opioid receptors in mice and may represent a new lead peptide for further investigation for the development of novel therapeutic approaches for depression.

Angiotensin (1-7) Attenuates the Nociceptive Behavior Induced by Substance P and NMDA via Spinal MAS1.

The intrathecal (i.t.) injection of substance P (SP) and N-methyl-D-aspartate (NMDA) induce transient nociceptive response by activating neurokinin (NK) 1 and NMDA receptors, respectively. We have recently reported that angiotensin (Ang) (1-7), an N-terminal fragment of Ang II, could alleviate several types of pain including neuropathic and inflammatory pain by activating spinal MAS1. Here, we investigated whether Ang (1-7) can inhibit the SP- and NMDA-induced nociceptive response. The nociceptive response induced by an i.t. injection of SP or NMDA was assessed by measuring the duration of hindlimb scratching directed toward the flank, biting and/or licking of the hindpaw or the tail for 5 min. Localization of MAS1 and either NK1 or NMDA receptors in the lumbar superficial dorsal horn was determined by immunohistochemical observation. The nociceptive response induced by SP and NMDA was attenuated by the i.t. co-administration of Ang (1-7) (0.03-3 pmol) in a dose-dependent manner. The inhibitory effects of Ang (1-7) (3 pmol) were attenuated by A779 (100 pmol), a MAS1 antagonist. Moreover, immunohistochemical analysis showed that spinal MAS1 co-localized with NK1 receptors and NMDA receptors on cells in the dorsal horn. Taken together, the i.t. injection of Ang (1-7) attenuated the nociceptive response induced by SP and NMDA via spinal MAS1, which co-localized with NK1 and NMDA receptors. Thus, the spinal Ang (1-7)/MAS1 pathway could represent a therapeutic target to effectively attenuate spinal pain transmission caused by the activation of NK1 or NMDA receptors.

Update of the GRIP web service.

G protein-coupled receptors (GPCRs) can form homodimers, heterodimers, or higher-order molecular complexes (oligomers). The reports on the change of functions through the oligomerization have been accumulated. Inhibition of GPCR oligomerization without affecting the protomer's overall structure would clarify the oligomer-specific functions although inhibition experiments are costly and require accurate information about the interface location. Unfortunately, the number of experimentally determined interfaces is limited. The precise prediction of the oligomerization interfaces is, therefore, useful for inhibition experiments to examine the oligomer-specific functions, which would accelerate investigations of the GPCR signaling. However, interface prediction for GPCR oligomerization is difficult because different GPCR subtypes belonging to the same subfamily often use different structural regions as their interfaces. We previously developed a high-performance method to predict the interfaces for GPCR oligomerization, by identifying the conserved surfaces with the sequence and structure information. Then, the structural characteristic of a GPCR structure is regarded to be a thick-tube like conformation that is approximately perpendicular to the membrane plane. Our method had successfully predicted all of the interfaces available on that day. We had launched a web server for our interface prediction of GPCRs (GRIP). We have improved the previous version of GRIP server and enhanced its usability. First, we discarded the approximation of the GPCR structure as the thick-tube-like conformation. This improvement increased the number of structures for the prediction. Second, the FUGUE-based template recommendation service was introduced to facilitate the choice of an appropriate structure for the prediction. The new prediction server is available at http://grip.b.dendai.ac.jp/∼grip/.

Liver hydrolysate improves depressive-like behavior in olfactory bulbectomized mice: Involvement of hippocampal neurogenesis through the AMPK/BDNF/CREB pathway.

Recently, we has reported that AMPK activator has antidepressant effect. Previous our study suggested that liver hydrolysate (LH) activated adenosine monophosphate-activated protein kinase (AMPK) in periphery. However, the effect of LH on depression is unclear. Therefore, we examines whether LH has antidepressant effect on olfactory bulbectomized (OBX) mice. OBX mice showed depressive-like behavior in tail-suspension test and reduction of hippocampal neurogenesis, while these changes were reversed by LH. LH enhanced hippocampal phosphate-AMPK, brain-derived neurotrophic factor (BDNF) and phosphate-cyclic adenosine monophosphate response element-binding protein (CREB) in OBX mice. These data indicate that LH may produce antidepressant effects via hippocampal AMPK/BDNF/CREB signaling.

Effect of Enterococcus faecalis 2001 on colitis and depressive-like behavior in dextran sulfate sodium-treated mice: involvement of the brain-gut axis.

BACKGROUND: Patients with inflammatory bowel disease (IBD), including those with ulcerative colitis and Crohn's disease, have higher rates of psychiatric disorders, such as depression and anxiety; however, the mechanism of psychiatric disorder development remains unclear. Mice with IBD induced by dextran sulfate sodium (DSS) in drinking water exhibit depressive-like behavior. The presence of Lactobacillus in the gut microbiota is associated with major depressive disorder. Therefore, we examined whether Enterococcus faecalis 2001 (EF-2001), a biogenic lactic acid bacterium, prevents DSS-induced depressive-like behavior and changes in peripheral symptoms. METHODS: We evaluated colon inflammation and used the tail suspension test to examine whether EF-2001 prevents IBD-like symptoms and depressive-like behavior in DSS-treated mice. The protein expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), X-linked inhibitor of apoptosis protein (XIAP), and cleaved caspase-3 in the rectum and hippocampus was assessed by western blotting. Hippocampal neurogenesis, altered nuclear factor-kappa B (NFκB) p65 morphometry, and the localization of activated NFκB p65 and XIAP were examined by immunohistochemistry. RESULTS: Treatment with 1.5% DSS for 7 days induced IBD-like pathology and depressive-like behavior, increased TNF-α and IL-6 expression in the rectum and hippocampus, activated caspase-3 in the hippocampus, and decreased hippocampal neurogenesis. Interestingly, these changes were reversed by 20-day administration of EF-2001. Further, EF-2001 administration enhanced NFκB p65 expression in the microglial cells and XIAP expression in the hippocampus of DSS-treated mice. CONCLUSION: EF-2001 prevented IBD-like pathology and depressive-like behavior via decreased rectal and hippocampal inflammatory cytokines and facilitated the NFκB p65/XIAP pathway in the hippocampus. Our findings suggest a close relationship between IBD and depression.

Antidepressant-like effect of aripiprazole via 5-HT1A, D1, and D2 receptors in the prefrontal cortex of olfactory bulbectomized mice.

Olfactory bulbectomized (OBX) mice exhibit depressive-like behaviors and memory deficits. We have reported that aripiprazole (ARI) ameliorates the behavioral hyper-responsivity to dopamine agonists and memory deficits in OBX mice; however, it is unclear whether ARI affects OBX-induced depressive-like behavior. To address this question, we evaluated the effect of ARI on depressive-like behavior in OBX mice using the forced swim test (FST). In addition, we investigated the effect of ARI on c-Fos expression in the prefrontal cortex (PFC), striatum, and hippocampus of OBX mice using western blotting. OBX mice exhibited a longer immobility duration in the FST 14 days after surgery. Depressive-like behavior in OBX mice was reversed 30 min after administration of ARI (0.01 or 0.03 mg/kg). In addition, c-Fos expression was increased in the PFC, but not the striatum or hippocampus, 30 min after acute administration of ARI. These effects were inhibited by administration of the selective 5-HT1A, D1, and D2 receptor antagonists, WAY100635, SCH23390, and L-741,626, respectively. These findings suggest that ARI produces an antidepressant effect in OBX mice that may be mediated by 5-HT1A, D1, and D2 receptors in the PFC.

Effect of repeated oral administration of chondroitin sulfate on neuropathic pain induced by partial sciatic nerve ligation in mice.

We examined whether chondroitin sulfate (CS), a compound used to treat osteoarthritis and joint pain, is effective against partial sciatic nerve ligation (PSNL)-induced neuropathic pain. Repeated oral administration of CS (300 mg/kg, b.i.d. for 20 days) resulted in inhibition of tactile allodynia observed 21 days after PSNL. On day 21, phosphorylation of spinal p38 mitogen-activated protein kinase (MAPK) was attenuated by CS. CS also inhibited c-Fos upregulation in ipsilateral deep dorsal horn (laminae III-IV) neurons, which receive Aß-fiber afferent inputs. These findings suggest that CS attenuates PSNL-induced tactile allodynia by inhibiting spinal p38 MAPK phosphorylation and Aß-fiber activation.

Kappa Opioid Receptor Agonist Administration in Olfactory Bulbectomized Mice Restores Cognitive Impairment through Cholinergic Neuron Activation.

Olfactory bulbectomized (OBX) mice are characterized by impaired performance in the passive avoidance test and decreased number of cholinergic neurons in the hippocampus. Several studies have reported that κ-opioid receptor agonists improve cognitive function in mice. However, their influence on OBX-induced cognitive dysfunction remains unclear. To address this question, we evaluated the effects of the endogenous κ-opioid receptor agonist dynorphin A (Dyn A) and the selective agonist trans-(-)-U-50488 on the behavior of OBX mice in the passive avoidance test. The cognitive dysfunction of OBX mice was significantly recovered by the intracerebroventricular administration of Dyn A or trans-(-)-U-50488. The effects of these two agonists were counteracted by the selective κ-opioid receptor antagonist nor-binaltorphimine or the inhibitor of acetylcholine release ß-bungarotoxin. These findings suggest that κ-opioid receptor agonists produce anti-dementia effects through activation of cholinergic neurons in OBX mice.

Collaborative environmental DNA sampling from petal surfaces of flowering cherry Cerasus × yedoensis 'Somei-yoshino' across the Japanese archipelago.

Recent studies have shown that environmental DNA is found almost everywhere. Flower petal surfaces are an attractive tissue to use for investigation of the dispersal of environmental DNA in nature as they are isolated from the external environment until the bud opens and only then can the petal surface accumulate environmental DNA. Here, we performed a crowdsourced experiment, the "Ohanami Project", to obtain environmental DNA samples from petal surfaces of Cerasus × yedoensis 'Somei-yoshino' across the Japanese archipelago during spring 2015. C. × yedoensis is the most popular garden cherry species in Japan and clones of this cultivar bloom simultaneously every spring. Data collection spanned almost every prefecture and totaled 577 DNA samples from 149 collaborators. Preliminary amplicon-sequencing analysis showed the rapid attachment of environmental DNA onto the petal surfaces. Notably, we found DNA of other common plant species in samples obtained from a wide distribution; this DNA likely originated from the pollen of the Japanese cedar. Our analysis supports our belief that petal surfaces after blossoming are a promising target to reveal the dynamics of environmental DNA in nature. The success of our experiment also shows that crowdsourced environmental DNA analyses have considerable value in ecological studies.

Involvement of Spinal Angiotensin II System in Streptozotocin-Induced Diabetic Neuropathic Pain in Mice.

Renin-angiotensin system (RAS) activity increases under hyperglycemic states, and is thought to be involved in diabetic complications. We previously demonstrated that angiotensin (Ang) II, a main bioactive component of the RAS, might act as a neurotransmitter and/or neuromodulator in the transmission of nociceptive information in the spinal cord. Here, we examined whether the spinal Ang II system is responsible for diabetic neuropathic pain induced by streptozotocin (STZ). Tactile allodynia was observed concurrently with an increase in blood glucose levels the day after mice received STZ (200 mg/kg, i.v.) injections. Tactile allodynia on day 14 was dose-dependently inhibited by intrathecal administration of losartan, an Ang II type 1 (AT1) receptor antagonist, but not by PD123319, an AT2 receptor antagonist. In the lumbar dorsal spinal cord, the expression of Ang II, Ang converting enzyme (ACE), and phospho-p38 mitogen-activated protein kinase (MAPK) were all significantly increased on day 14 after STZ injection compared with vehicle-treated controls, whereas no differences were observed among AT1 receptors or angiotensinogen levels. Moreover, the increase in phospho-p38 MAPK was significantly inhibited by intrathecal administration of losartan. These results indicate that the expression of spinal ACE increased in STZ-induced diabetic mice, which in turn led to an increase in Ang II levels and tactile allodynia. This increase in spinal Ang II was accompanied by the phosphorylation of p38 MAPK, which was shown to be mediated by AT1 receptors.

GGIP: Structure and sequence-based GPCR-GPCR interaction pair predictor.

G Protein-Coupled Receptors (GPCRs) are important pharmaceutical targets. More than 30% of currently marketed pharmaceutical medicines target GPCRs. Numerous studies have reported that GPCRs function not only as monomers but also as homo- or hetero-dimers or higher-order molecular complexes. Many GPCRs exert a wide variety of molecular functions by forming specific combinations of GPCR subtypes. In addition, some GPCRs are reportedly associated with diseases. GPCR oligomerization is now recognized as an important event in various biological phenomena, and many researchers are investigating this subject. We have developed a support vector machine (SVM)-based method to predict interacting pairs for GPCR oligomerization, by integrating the structure and sequence information of GPCRs. The performance of our method was evaluated by the Receiver Operating Characteristic (ROC) curve. The corresponding area under the curve was 0.938. As far as we know, this is the only prediction method for interacting pairs among GPCRs. Our method could accelerate the analyses of these interactions, and contribute to the elucidation of the global structures of the GPCR networks in membranes. Proteins 2016; 84:1224-1233. © 2016 Wiley Periodicals, Inc.

Chondroitin sulfate attenuates formalin-induced persistent tactile allodynia.

We examined the effect of chondroitin sulfate (CS), a compound used in the treatment of osteoarthritis and joint pain, on the formalin-induced tactile allodynia in mice. A repeated oral administration of CS (300 mg/kg, b.i.d.) significantly ameliorated the formalin-induced tactile allodynia from day 10 after formalin injection. On day 14, the phosphorylation of spinal p38 MAPK and subsequent increase in c-Fos-immunoreactive dorsal lumbar neurons were attenuated by the repeated administration of CS. These findings suggest that CS attenuates formalin-induced tactile allodynia through the inhibition of p38 MAPK phosphorylation and subsequent up-regulation of c-Fos expression in the dorsal lumbar spinal cord.

The Bisphosphonates Clodronate and Etidronate Exert Analgesic Effects by Acting on Glutamate- and/or ATP-Related Pain Transmission Pathways.

Bisphosphonates (BPs) are typical anti-bone-resorptive drugs, with nitrogen-containing BPs (N-BPs) being stronger than non-nitrogen-containing BPs (non-N-BPs). However, N-BPs have inflammatory/necrotic effects, while the non-N-BPs clodronate and etidronate lack such side effects. Pharmacological studies have suggested that clodronate and etidronate can (i) prevent the side effects of N-BPs in mice via inhibition of the phosphate transporter families SLC20 and/or SLC34, through which N-BPs enter soft-tissue cells, and (ii) also inhibit the phosphate transporter family SLC17. Vesicular transporters for the pain transmitters glutamate and ATP belong to the SLC17 family. Here, we examined the hypothesis that clodronate and etidronate may enter neurons through SLC20/34, then inhibit SLC17-mediated transport of glutamate and/or ATP, resulting in their decrease, and thereby produce analgesic effects. We analyzed in mice the effects of various agents [namely, intrathecally injected clodronate, etidronate, phosphonoformic acid (PFA; an inhibitor of SLC20/34), and agonists of glutamate and ATP receptors] on the nociceptive responses to intraplantar injection of capsaicin. Clodronate and etidronate produced analgesic effects, and these effects were abolished by PFA. The analgesic effects were reduced by N-methyl-D-aspartate (agonist of the NMDA receptor, a glutamate receptor) and α,ß-methylene ATP (agonist of the P2X-receptor, an ATP receptor). SLC20A1, SLC20A2, and SLC34A1 were detected within the mouse lumbar spinal cord. Although we need direct evidence, these results support the above hypothesis. Clodronate and etidronate may be representatives of a new type of analgesic drug. Such drugs, with both anti-bone-resorptive and unique analgesic effects without the adverse effects associated with N-BPs, might be useful for osteoporosis.

Liver hydrolysate attenuates the sickness behavior induced by concanavalin A in mice.

Liver hydrolysate (LH) is used as a pharmaceutical agent in Japan, to enhance liver function. However, the effects of LH on sickness behavior are unknown. This study investigated the effect of LH on sickness behaviors, such as concanavalin A (ConA)-induced reduction of locomotor activity. ConA treatment significantly decreased locomotor activity. The striatal tyrosine hydroxylase (TH) levels were also significantly decreased following ConA treatment. The decreased locomotor activity and TH levels were significantly reversed by LH treatment. LH may prove beneficial for preventing sickness behavior following ConA treatment, at least in part, by activating TH in the striatum.

Clinical and preclinical evidence that angiotensin-converting enzyme inhibitors and angiotensin receptor blockers prevent diabetic peripheral neuropathy.

Given possible involvement of the central and peripheral angiotensin system in pain processing, we conducted clinical and preclinical studies to test whether pharmacological inhibition of the angiotensin system would prevent diabetic peripheral neuropathy (DPN) accompanying type 2 diabetes mellitus (T2DM). In the preclinical study, the nociceptive sensitivity was determined in leptin-deficient ob/ob mice, a T2DM model. A clinical retrospective cohort study was conducted, using the medical records of T2DM patients receiving antihypertensives at three hospitals for nearly a decade. In the ob/ob mice, daily treatment with perindopril, an angiotensin-converting enzyme inhibitor (ACEI), or telmisartan, an angiotensin receptor blocker (ARB), but not amlodipine, an L-type calcium channel blocker (CaB), significantly inhibited DPN development without affecting the hyperglycemia. In the clinical study, the enrolled 7464 patients were divided into three groups receiving ACEIs, ARBs and the others (non-ACEI, non-ARB antihypertensives). Bonferroni's test indicated significantly later DPN development in the ARB and ACEI groups than the others group. The multivariate Cox proportional analysis detected significant negative association of the prescription of ACEIs or ARBs and ß-blockers, but not CaBs or diuretics, with DPN development. Thus, our study suggests that pharmacological inhibition of the angiotensin system is beneficial to prevent DPN accompanying T2DM.