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Minocycline attenuates the development of diabetic neuropathy by inhibiting spinal cord Notch signaling in rat
Biomedicine & Pharmacotherapy 94 (2017) 380–385
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Minocycline attenuates the development of diabetic neuropathy by inhibiting spinal cord Notch signaling in rat Cheng Yangb,1, Jie Gaob,1, Banglin Wuc , Nuo Yana , Hui Lia , Yiqing Rena , Yufei Kand, Jiamin Liange, Yang Jiaof , Yonghao Yuf,* a
Department of Anesthesiology, Affiliated Hospital of Logistics University of PAP, Tianjin, 300162, China Tianjin Medical University Graduate School, Department of Anesthesiology, Affiliated Hospital of logistics University of PAP, Tianjin, 300162, China The Central Hospital of Enshi Autonomous Prefecture, Enshi, 445000, China d TianJin Medical University, Tianjin, 300070, China e Operating Room, Affiliated Hospital of Logistics University of PAP, 300162, China f Department of Anesthesiology, General Hospital of Tianjin Medical University, Tianjin, 300052, China b c
A R T I C L E I N F O
Article history: Received 4 January 2017 Received in revised form 14 July 2017 Accepted 18 July 2017 Keywords: Notch signaling Minocycline DNP Thermal pain threshold DAPT
A B S T R A C T
We studied the effects of minocycline (an inhibitor of microglial activation) on the expression and activity of Notch-1 receptor, and explored the therapeutic efficacy of minocycline combined with Notch inhibitor DAPT in the treatment of diabetic neuropathic pain (DNP). Diabetic rat model was established by intraperitoneal injection (ip) of Streptozotocin (STZ). Expression and activity of Notch-1 and expression of macrophage/microglia marker Iba-1 were detected by WB. Diabetes induction significantly attenuated sciatic nerve conduction velocity, and dramatically augmented the expression and the activity of Notch-1 in the lumbar enlargement of the spinal cord. Minocycline treatment, however, accelerated the decreased conduction velocity of sciatic nerve and suppressed Notch-1expression and activity in diabetic rats. Similar to DAPT treatment, minocycline administration also prolonged thermal withdrawal latency (TWL) and increase mechanical withdrawal threshold (MWT) in diabetic rats in response to heat or mechanical stimulation via inhibition the expression and the activity of Notch-1 in spinal cord. Combination of DAPT and minocycline further inhibited Notch-1 receptor signaling and reduce neuropathic pain exhibited as improved TWL and MWT. Our study revealed a novel mechanism of Notch1 receptor inhibition in spinal cord induced by minocycline administration, and suggested that the combination of minocycline and DAPT has the potential to treat DNP. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Diabetes mellitus (DM) is a chronic progressive metabolic disorder characterized by hyperglycemia, mainly due to inadequate insulin secretion or resistance [1–3]. Long-term hyperglycemia leads to the pathogenesis of diabetic neuropathy and other complications via multiple mechanisms [1,3]. Identification of the signaling pathways underlying these mechanisms would be
Abbreviations: DNP, diabetic neuropathic pain; Ip, intraperitoneal injection; STZ, streptozotocin; TWL, thermal withdrawal latency; MWT, mechanical withdrawal threshold; DM, diabetes mellitus; DRG, dorsal root ganglion; CNS, central nerve system; WB, western blot; NICD, notch intracellular domain. * Corresponding author at: Department of Anesthesiology, General Hospital of Tianjin Medical University, 154 AnShan Road, Heping District of Tianjin, 300052, China. E-mail address: [email protected] (Y. Yu). 1 Cheng Yang and Jie Gao contribute equally to this work. http://dx.doi.org/10.1016/j.biopha.2017.07.078 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
beneficial in exploiting the pharmacological approaches to inhibit or activate relevant signaling molecules, and hold great promise to treat DM and its complications. Diabetic neuropathy is a common complication, usually manifested as spontaneous pain, hyperalgesia, allodynia, parasthesias and dysthesias that occur more in the distal limb symmetrically [4,5]. Accumulating evidence suggests that about 50% of diabetic patients eventually develop into diabetic neuropathy [6] that is linked to depression, suicidal tendencies and foot amputation [7]. The mechanisms underlying DNP is complicated that involve hyper-excitability in the affected dorsal root ganglion (DRG) neurons, aberrant myelination, alterations in synaptic plasticity and excitatory and inhibitory mechanisms in spinal cord, et al. [7– 10]. Despite significant improvement in treatment, DNP frequently remains unresponsive to most regimens [3]. Highly conserved Notch signaling determines cell fate in developing spinal cord and
C. Yang et al. / Biomedicine & Pharmacotherapy 94 (2017) 380–385
plays keys roles in synaptic plasticity in adult central nerve system (CNS) [11]. Activation of Notch signaling promotes the generation of excitatory neurons from the sensory interneuron progenitors [11]. And the expression and activity of notch family members are significantly augmented after nerve injury [12]. Moreover, Notch signaling is a major determinant of precursor cells development to neurons or glia. Activation of Notch pathway promoted the identity of radial glial and maintained their proliferative state [13]. Notch activation also promoted Muller glia formation in retina [14], and astrocytes generation in adult hippocampus [15]. Recent studies also show that Notch signaling is critical to the development of neuropathic pain [16] and diabetic nephropathy including glomerulopathy, tubulointerstitial fibrosis, and possibly arteriopathy and inflammation [17]. However, the function of Notch pathway in diabetes-induced neuropathy has not been widely explored yet. Traditional opinion of neuropathic pain is primarily raised from dysfunctional or damaged nerve fibers of neurons. However, new evidence suggested that microglia and other non-neuron cells plays key roles in this process. Activated microglia release a variety of neuromodulators, neuroactive substances, and proinflammatory cytokines that have directly participated in the neuropathic pain induction [18–21]. Minocycline attenuated the development of DNP possible via anti-inflammatory and antioxidant mechanisms. However, little is known about the role of Notch signaling in microglia-mediated DNP. In the present studies, we addressed the issue and uncovered a novel mechanism of Notch-1 receptor signaling-mediated microglia activation and demonstrated the synergistic effect of microglia inhibition plus Notch blockage in attenuation of DNP. 2. Materials and methods 2.1. Experimental animals To study the effects of minocycline (an inhibitor of microglial activation) on the expression and activity of Notch-1 receptor, 40 SD rats weighing 180–220 g were randomly divided into 4 groups (10 for each): control group (C group), minocycline treated group (Mino group), diabetic group (DM group), and diabetic group treated with minocycline (DM + Mino group). Diabetic rat model was established by intraperitoneal injection (ip) of single dose Streptozotocin (STZ), 65 mg/Kg[22]. Minocycline treatment started two weeks after STZ ip. To investigate the synergistic effect of minocycline with DAPT in treating DNP, another 40 SD rats weighing 180–220 g was randomly divide into 4 groups with 10 rats in each group: DM group, DAPT group, Mino group, and DAPT + Mino group. STZ was injected to all rats to induce diabetes first. DAPT ip was conducted 3 times per week afterwards. Minocycline was given 2 weeks after diabetes induction. Thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) of the rats were measured weekly. The expressions of Notch-1, Iba-1, NICD and Hes-1 in spinal cord (L4-L6) were detected by WB at the end of the experiment (week 8). The rats were housed under standard laboratory conditions and allowed food and water ad libitum. The animal care and experiment protocols were approved by the Animal Care Committee and Institutional Biosafety Committee of Affiliated Hospital of logistics University of PAP.
2.2. Induction of diabetes [23] SD rats were rendered diabetic with single dose of 65 mg/kg STZ ip with about 10% mortality. Diabetes was confirmed by
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measurement of tail vein blood glucose concentration weekly by portable personal blood glucose meter. Only rats with blood glucose level >16.7 mM were included in the study. 2.3. Reagents and materials STZ (S0130) and DAPT (D5942) was purchased from SigmaAldrich; and minocycline hydrochloride (M2288) was from Tokyo Chemical Industry Co., Ltd.; Notch-1 (4380) and Hes-1 monoclonal antibodies were purchased from CST (USA); rabbit anti-rat NICD and anti-Iba-1 (ab15691) polyclonal antibodies as well as mouse anti-rat Histon-1 antibody were from Abcam; and anti-b-actin (AP0060)polyclonal antibody were from Epitomics. 2.4. Behavioral studies 2.4.1. Measurement of the sural nerve conduction velocity [24,25] Animals were habituated to the testing environment daily for 3 days before baseline testing. The rats under anesthesia (10% Chloral hydrate, 1 ml/100 mg body weight, ip) were placed in prone position, and the airway was kept unobstructed. The stimulating electrodes were positioned between the femur and the calcaneal tubercle. The recording electrodes were positioned subcutaneous of the ankle where the sciatic nerve go through. Occasionally the right sciatic nerves were exposed through a dorsolateral incision in case the signal could not be recorded correctly, and the sural nerve conduction velocity was recorded through an automatic potentiometric analysis system evoked by Viking Quest electromyography. The sciatic nerve was stimulated with bipolar needle electrodes placed on sciatic notch between the femur and the Ischial tuberosity, and recording was done on ankle where the sciatic nerve passes. Electric stimulus were delivered at 2 Hz with the voltage of 1–3 V and the currency of 2.5-3.5 mV. The latency of the evoked muscle twitch was recorded from the intrinsic foot muscles with bipolar needle electrode. The distance between the two points of stimulating electrodes on the skin and latencies of action potentials were measured. Conduction velocity was calculated using the following formula. Sural nerve conduction velocity = Dd/Dt, in which the sural nerve conduction velocity is motor nerve conduction velocity (cm/ms), Dd is the distances between stimulatory and recording electrodes, and Dt is the difference between delay times of recorded compound action potentials at points of two electrodes. 2.4.2. Assessment of TWL [26] The rats were placed in a hot plexiglass plate with the temperature maintained at 52.0 0.5 C until the signs of struggle such as licking feet, crying and leaping were observed. The baseline TWL was established by averaging 3 experiment results recorded with 10 min apart. A cut-off time of 15 s was imposed to avoid injury to the rats. 2.4.3. Measurement of mechanical stimulated-pain threshold [26] The rats were placed in the mesh plane to adapt to the experiment environment for at least 10 min, and were then connect to the Von Frey electronic measuring instrument with the pressure detecting sensor. The rigid plastic probe was installed on the handle, and the pressure was gradually increased to the hind feet of the rats. The mechanical threshold (g) was recorded as the rats appeared response, and the mean value was taken as the mechanical pain threshold (MWT). Triple experiments were conducted for each rats with 10 min interval. The experiment results would be excluded if the pressure exceeded 500 g.
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Table 1 Comparisonof glucose concentration among different groups (mM, n = 10, Mean SD). Group
T1
T2
T3
T4
C Mino DM DM + Mino
5.45 0.87 5.28 0.68 5.31 0.72 5.38 1.14
6.33 0.99 5.63 0.89 19.41 3.27* 20.04 2.97*
5.76 0.71 4.63 0.92 21.04 2.40* 22.00 3.44*
5.11 0.32 5.37 0.56 23.04 3.43* 21.35 3.53*
The experiment was carried out before treatment (T1), 2 weeks (T2), 4 weeks (T3) and 8 weeks (T4) post treatment. * P < 0.01 vs Group C.
2.5. Detection of notch-1, iba-1, NICD and hes-1 expression in spinal cord by WB 8 weeks post diabetes induction, the lumbar enlargement (L4 L6) of the spinal cord was retrieved from anesthetized rats and snap frozen in liquid nitrogen. The total protein was extracted, quantified, prepared for SDS-PAGE, and detect by WB using indicated antibodies. The optical density of the bands on the film was analyzed by image analysis system under the chemiluminescence environment. The relative expression level of target proteins was compared to internal reference b-actin or Histon-1.
Table 2 Comparison of body weight among different groups (g, n = 10, Mean SD). Group
T1
T2
T3
T4
C Mino DM DM + Mino
198.28 7.85 197.12 10.54 196.76 2.33 203.20 10.14
243.72 14.52 226.08 12.80 183.44 14.66* 204.16 11.81
305.32 16.17 303.18 15.52 175.32 16.99* 199.64 18.29**
351.96 5.92 355.20 7.77 153.10 15.42* 220.10 15.99**
The body weight was measured before treatment (T1), 2 weeks (T2), 4 weeks (T3) and 8 weeks (T4) post treatment. * P < 0.01 vs Group C. ** P < 0.05 vs Group DM.
Table 3 Minocycline administration improved sural nerve conduction velocity (unit: m/s) in diabetic rats. Group
T1
T2
T3
T4
C Mino DM DM + Mino
50.74 5.31 53.83 10.91 52.06 9.26 51.15 14.76
51.31 5.88 48.29 6.39 35.58 8.09* 39.78 7.77*
50.83 10.74 49.59 5.17 25.32 5.24* 41.02 8.68*~
51.39 10.74 51.13 6.58 24.43 4.70* 44.67 3.45~
Note: compared with C group, *P < 0.01; compared with DM group, ~P < 0.05; The experiment was carried out before treatment (T1), 2 weeks (T2), 4 weeks (T3) and 8 weeks (T4) post treatment.
2.6. Minocycline and DAPT administration Two weeks after diabetes induction, minocycline was dissolved in 0.9% saline and administered ip to both Mino group and DM+ Mino group rats at a dose of 40 mg/kg each day for 3 days. The rats in C group and DM group were given the same volume of saline ip as vehicle control. The sural nerve conduction velocity was measured before treatment (T1), 2 weeks (T2), 4 weeks (T3) and 8 weeks (T4) post treatment. The TWL and MWT were measured weekly (W0-W8) during the same period of time, in which W0
refer to the baseline value undertook before treatment. At week 8, the rats were sacrificed and the Notch-1, Iba-1, NICD and Hes-1 expression were detected by WB in the lumbar enlargement of spinal cord (L4 L6). To study the synergistic effect of minocycline and DAPT on the treatment of DNP, another 40 rats were subjected to STZ administration to induce diabetes and divide into four groups (10 rats/group): DM group, DAPT group, Mino group, and DAPT + Mino group. DAPT (1 mg/kg) was administrated by ip 3 times/
Fig. 1. Minocycline administration significant inhibited Notch-1 expression and activity in diabetic rats. A and C: Male adults SD rats were rendered diabetic and treated with 40 mg/kg minocycline for 2 weeks. The expression of Notch-1 receptor, Iba-1 (A), NICD, and Hes-1 (B) in lumber spinal cord were assessed by WB with indicated antibodies 8 weeks post diabetes induction. B and D: Quantification of the protein levels of Notch-1, Iba-1 (B), NICD, and Hes-1 (D) in (A) and (C). * P < 0.05, ** P < 0.01, *** P < 0.001).
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week. Minocycline was given as above mentioned. The TWL and MWT in each rat were measured from before STZ injection (T0) to the next 8 week (W1-W8). The rats were sacrificed at end time point (W8), and the spinal cord of L4-L6 was collected and subject for WB. 2.7. Statistical analysis The WB bands were scanned and quantified by Quantity One Image Analysis System. The data were presented as mean standard deviation (SD), and analyzed by One-way ANOVA (SPSS 17.0 software). P < 0.05 was considered as statistically significant. 3. Results 3.1. Minocycline treatment accelerated sural nerve conduction velocity in diabetic rats via inhibition notch-1 expression and activity The dramatically increase blood glucose concentration (Table. 1) and attenuated body weight (Table. 2) in DM group suggested that the SD rats were rendered diabetic post 65 mg/kg STZ ip. The sural nerve conduction velocity of diabetic rats was significantly decreased 2 weeks post STZ injection (T2) and continued
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decreasing till 8 weeks (T4) compared to non-diabetic rats (C group) (Table. 3), which is in line with our previously published data[27]. Administration of minocycline in diabetic rats significantly accelerated the attenuated nerve conduction compare with saline vehicle controls (DM group) (Table. 3), suggesting that minocycline is effective in improving the conduction of sciatic nerve in diabetes rats. Consistent with these results, the expression of Notch-1 receptor as well as the receptor activation product NICD and the downstream target Hes-1 was significantly augmented in the luminal spinal cord of STZ-induced diabetic rats (Fig. 1A and D). The expression of macrophage/microglia marker Iba-1 was also upregulated in diabetic mice (Fig. 1A and B), indicating the presence of inflammation and microglia proliferation. Administration of minocycline, however, dramatically blocked Notch-1 expression and activation (Fig. 1 and 2), as well as the expression of Iba-1. 3.2. Administration of minocycline attenuated TWL and MWT in diabetic rats through notch inhibition The threshold for thermal and mechanical hyperalgesia presented as TWL and MWT were gradually decreased in STZinduced diabetic rats (Fig. 2A and B). Chronic administration of
Fig. 2. The combination of Minocycline with DAPT significant alleviated DNP via inhibition the expression and activity of Notch-1. A and B: Male adult SD rats were subjected to STZ to induce diabetes. 40 mg/kg minocycline or DAPT (1 mg/kg) was given alone or in combination. TWL and MWT were recorded weekly for 8 weeks post STZ injection. Mean SD; n = 10/group. Compared with DM group, * p < 0.05, ** p < 0.01.C and E: 8 weeks after STZ induction, the lumber spinal cord (L4-L6) was retrieved and subjected to WB using indicated antibodies. D and F: Quantification of the protein expression of Notch-1, Iba-1 (D), NICD, and Hes-1 (F) in (C) and (E). * P < 0.05, ** P < 0.01.
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Notch inhibitor DAPT or minocycline for 2 weeks markedly prevented the development of thermal and mechanical hyperalgesia (Fig. 2A and B), and inhibited the expression and activity of Notch-1that were exhibited as significantly attenuated expression of Notch-1, NICD, and Hes-1 (Fig. 1, Fig. 2C and F). The expression of Iba-1 was also downregulated (Fig. 2C), indicating that minocycline suppressed Notch-1-dependent inflammation and microglia proliferation in spinal cord. Furthermore, the combination of minocycline and DAPT synergistically improved TWL and MWT (Fig. 2A and B) and inhibited Notch-1 expression and activity (Fig. 2C and F). Thus our studies unrevealed a novel mechanism of minocycline in treatment DNP via blocking Notch signaling and suggested the new regimen by combination of minocycline and DAPT holds big promise in the care and management of diabetes and its complications. 4. Discussion Here, we have identified an important role of Notch-1 in microglia in mediating the development of diabetic neuropathy. Inhibition of Notch-1expression and activity by DAPT and/or minocycline inhibited the microglia proliferation and alleviated thermal and mechanical hyperalgesia that frequently manifested in DNP. Activation of Notch-1-mediated Notch signaling pathway depends on the interaction of Notch-1-expressing cells with neighboring cells that express Notch-1 activators such as Jagged or Delta [28]. Ligand binding to Notch-1 induces the proteolytic release by the c-secretase of the Notch intracellular domain (NICD) that trans-locates to the nucleus to regulate the expression of specific target genes such as HES-1 and HEY-1 [28–30]. We have observed that not only the expression of Notch-1, but also the NICD of Notch-1 receptor as well as the downstream Notch target Hes-1 was augmented post STZ administration, indicating that Notch activity was enhanced in diabetic neuropathy. Our discovery is consistent with the previous finding that Notch signaling is activated during nerve injury [12,16], and inactivation of Notch signal transduction pathway before or after the appearance of pain sensitivity either delayed or prevented the progress of injury-induced neuropathy [16]. The biological effect of Notch activation is cellular context- and tissue environment-dependent. Notch-1 has been shown to play a fundamental role in regulating the fate of neural progenitor’s differentiation and survival in embryonic CNS [31,32]. However, recent findings also indicate that Notch-1 signal guides the commitment of neural progenitors to the glial cell fate [15,33,34]. Giving the fact that microglia and other non-neuron cells plays key roles in sensing neuropathic pain[35,36], it is reasonable to speculate that Notch signaling in microglia might have participated the initiation, development, or the progress of DNP. Thus the focus of our study was to explore Notch function in microglia using the specific microglia inhibitor minocycline. The underlying mechanisms of minocycline-mediated suppression of neuropathic pain include attenuating pro-inflammatory cytokines release and alleviating oxidative and nitrosative stress [35,37], and preventing impaired glial glutamate uptake in the spinal sensory synapses [38], et al. We demonstrated in this study that minocycline ameliorated DNP via down-regulation Notch expression and inactivation of Notch signaling in microglia within spinal cord. In addition, administration of minocycline with Notch inhibitor DAPT further strengthened the therapeutic effect of BAPT in treat diabetic neuropathy. Thus our study discovered an important role of Notch in microglia and suggested this novel regimen has the potential in treating DNP.
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Minocycline attenuates the development of diabetic neuropathy by inhibiting spinal cord Notch signaling in rat Cheng Yangb,1, Jie Gaob,1, Banglin Wuc , Nuo Yana , Hui Lia , Yiqing Rena , Yufei Kand, Jiamin Liange, Yang Jiaof , Yonghao Yuf,* a
Department of Anesthesiology, Affiliated Hospital of Logistics University of PAP, Tianjin, 300162, China Tianjin Medical University Graduate School, Department of Anesthesiology, Affiliated Hospital of logistics University of PAP, Tianjin, 300162, China The Central Hospital of Enshi Autonomous Prefecture, Enshi, 445000, China d TianJin Medical University, Tianjin, 300070, China e Operating Room, Affiliated Hospital of Logistics University of PAP, 300162, China f Department of Anesthesiology, General Hospital of Tianjin Medical University, Tianjin, 300052, China b c
A R T I C L E I N F O
Article history: Received 4 January 2017 Received in revised form 14 July 2017 Accepted 18 July 2017 Keywords: Notch signaling Minocycline DNP Thermal pain threshold DAPT
A B S T R A C T
We studied the effects of minocycline (an inhibitor of microglial activation) on the expression and activity of Notch-1 receptor, and explored the therapeutic efficacy of minocycline combined with Notch inhibitor DAPT in the treatment of diabetic neuropathic pain (DNP). Diabetic rat model was established by intraperitoneal injection (ip) of Streptozotocin (STZ). Expression and activity of Notch-1 and expression of macrophage/microglia marker Iba-1 were detected by WB. Diabetes induction significantly attenuated sciatic nerve conduction velocity, and dramatically augmented the expression and the activity of Notch-1 in the lumbar enlargement of the spinal cord. Minocycline treatment, however, accelerated the decreased conduction velocity of sciatic nerve and suppressed Notch-1expression and activity in diabetic rats. Similar to DAPT treatment, minocycline administration also prolonged thermal withdrawal latency (TWL) and increase mechanical withdrawal threshold (MWT) in diabetic rats in response to heat or mechanical stimulation via inhibition the expression and the activity of Notch-1 in spinal cord. Combination of DAPT and minocycline further inhibited Notch-1 receptor signaling and reduce neuropathic pain exhibited as improved TWL and MWT. Our study revealed a novel mechanism of Notch1 receptor inhibition in spinal cord induced by minocycline administration, and suggested that the combination of minocycline and DAPT has the potential to treat DNP. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Diabetes mellitus (DM) is a chronic progressive metabolic disorder characterized by hyperglycemia, mainly due to inadequate insulin secretion or resistance [1–3]. Long-term hyperglycemia leads to the pathogenesis of diabetic neuropathy and other complications via multiple mechanisms [1,3]. Identification of the signaling pathways underlying these mechanisms would be
Abbreviations: DNP, diabetic neuropathic pain; Ip, intraperitoneal injection; STZ, streptozotocin; TWL, thermal withdrawal latency; MWT, mechanical withdrawal threshold; DM, diabetes mellitus; DRG, dorsal root ganglion; CNS, central nerve system; WB, western blot; NICD, notch intracellular domain. * Corresponding author at: Department of Anesthesiology, General Hospital of Tianjin Medical University, 154 AnShan Road, Heping District of Tianjin, 300052, China. E-mail address: [email protected] (Y. Yu). 1 Cheng Yang and Jie Gao contribute equally to this work. http://dx.doi.org/10.1016/j.biopha.2017.07.078 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
beneficial in exploiting the pharmacological approaches to inhibit or activate relevant signaling molecules, and hold great promise to treat DM and its complications. Diabetic neuropathy is a common complication, usually manifested as spontaneous pain, hyperalgesia, allodynia, parasthesias and dysthesias that occur more in the distal limb symmetrically [4,5]. Accumulating evidence suggests that about 50% of diabetic patients eventually develop into diabetic neuropathy [6] that is linked to depression, suicidal tendencies and foot amputation [7]. The mechanisms underlying DNP is complicated that involve hyper-excitability in the affected dorsal root ganglion (DRG) neurons, aberrant myelination, alterations in synaptic plasticity and excitatory and inhibitory mechanisms in spinal cord, et al. [7– 10]. Despite significant improvement in treatment, DNP frequently remains unresponsive to most regimens [3]. Highly conserved Notch signaling determines cell fate in developing spinal cord and
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plays keys roles in synaptic plasticity in adult central nerve system (CNS) [11]. Activation of Notch signaling promotes the generation of excitatory neurons from the sensory interneuron progenitors [11]. And the expression and activity of notch family members are significantly augmented after nerve injury [12]. Moreover, Notch signaling is a major determinant of precursor cells development to neurons or glia. Activation of Notch pathway promoted the identity of radial glial and maintained their proliferative state [13]. Notch activation also promoted Muller glia formation in retina [14], and astrocytes generation in adult hippocampus [15]. Recent studies also show that Notch signaling is critical to the development of neuropathic pain [16] and diabetic nephropathy including glomerulopathy, tubulointerstitial fibrosis, and possibly arteriopathy and inflammation [17]. However, the function of Notch pathway in diabetes-induced neuropathy has not been widely explored yet. Traditional opinion of neuropathic pain is primarily raised from dysfunctional or damaged nerve fibers of neurons. However, new evidence suggested that microglia and other non-neuron cells plays key roles in this process. Activated microglia release a variety of neuromodulators, neuroactive substances, and proinflammatory cytokines that have directly participated in the neuropathic pain induction [18–21]. Minocycline attenuated the development of DNP possible via anti-inflammatory and antioxidant mechanisms. However, little is known about the role of Notch signaling in microglia-mediated DNP. In the present studies, we addressed the issue and uncovered a novel mechanism of Notch-1 receptor signaling-mediated microglia activation and demonstrated the synergistic effect of microglia inhibition plus Notch blockage in attenuation of DNP. 2. Materials and methods 2.1. Experimental animals To study the effects of minocycline (an inhibitor of microglial activation) on the expression and activity of Notch-1 receptor, 40 SD rats weighing 180–220 g were randomly divided into 4 groups (10 for each): control group (C group), minocycline treated group (Mino group), diabetic group (DM group), and diabetic group treated with minocycline (DM + Mino group). Diabetic rat model was established by intraperitoneal injection (ip) of single dose Streptozotocin (STZ), 65 mg/Kg[22]. Minocycline treatment started two weeks after STZ ip. To investigate the synergistic effect of minocycline with DAPT in treating DNP, another 40 SD rats weighing 180–220 g was randomly divide into 4 groups with 10 rats in each group: DM group, DAPT group, Mino group, and DAPT + Mino group. STZ was injected to all rats to induce diabetes first. DAPT ip was conducted 3 times per week afterwards. Minocycline was given 2 weeks after diabetes induction. Thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) of the rats were measured weekly. The expressions of Notch-1, Iba-1, NICD and Hes-1 in spinal cord (L4-L6) were detected by WB at the end of the experiment (week 8). The rats were housed under standard laboratory conditions and allowed food and water ad libitum. The animal care and experiment protocols were approved by the Animal Care Committee and Institutional Biosafety Committee of Affiliated Hospital of logistics University of PAP.
2.2. Induction of diabetes [23] SD rats were rendered diabetic with single dose of 65 mg/kg STZ ip with about 10% mortality. Diabetes was confirmed by
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measurement of tail vein blood glucose concentration weekly by portable personal blood glucose meter. Only rats with blood glucose level >16.7 mM were included in the study. 2.3. Reagents and materials STZ (S0130) and DAPT (D5942) was purchased from SigmaAldrich; and minocycline hydrochloride (M2288) was from Tokyo Chemical Industry Co., Ltd.; Notch-1 (4380) and Hes-1 monoclonal antibodies were purchased from CST (USA); rabbit anti-rat NICD and anti-Iba-1 (ab15691) polyclonal antibodies as well as mouse anti-rat Histon-1 antibody were from Abcam; and anti-b-actin (AP0060)polyclonal antibody were from Epitomics. 2.4. Behavioral studies 2.4.1. Measurement of the sural nerve conduction velocity [24,25] Animals were habituated to the testing environment daily for 3 days before baseline testing. The rats under anesthesia (10% Chloral hydrate, 1 ml/100 mg body weight, ip) were placed in prone position, and the airway was kept unobstructed. The stimulating electrodes were positioned between the femur and the calcaneal tubercle. The recording electrodes were positioned subcutaneous of the ankle where the sciatic nerve go through. Occasionally the right sciatic nerves were exposed through a dorsolateral incision in case the signal could not be recorded correctly, and the sural nerve conduction velocity was recorded through an automatic potentiometric analysis system evoked by Viking Quest electromyography. The sciatic nerve was stimulated with bipolar needle electrodes placed on sciatic notch between the femur and the Ischial tuberosity, and recording was done on ankle where the sciatic nerve passes. Electric stimulus were delivered at 2 Hz with the voltage of 1–3 V and the currency of 2.5-3.5 mV. The latency of the evoked muscle twitch was recorded from the intrinsic foot muscles with bipolar needle electrode. The distance between the two points of stimulating electrodes on the skin and latencies of action potentials were measured. Conduction velocity was calculated using the following formula. Sural nerve conduction velocity = Dd/Dt, in which the sural nerve conduction velocity is motor nerve conduction velocity (cm/ms), Dd is the distances between stimulatory and recording electrodes, and Dt is the difference between delay times of recorded compound action potentials at points of two electrodes. 2.4.2. Assessment of TWL [26] The rats were placed in a hot plexiglass plate with the temperature maintained at 52.0 0.5 C until the signs of struggle such as licking feet, crying and leaping were observed. The baseline TWL was established by averaging 3 experiment results recorded with 10 min apart. A cut-off time of 15 s was imposed to avoid injury to the rats. 2.4.3. Measurement of mechanical stimulated-pain threshold [26] The rats were placed in the mesh plane to adapt to the experiment environment for at least 10 min, and were then connect to the Von Frey electronic measuring instrument with the pressure detecting sensor. The rigid plastic probe was installed on the handle, and the pressure was gradually increased to the hind feet of the rats. The mechanical threshold (g) was recorded as the rats appeared response, and the mean value was taken as the mechanical pain threshold (MWT). Triple experiments were conducted for each rats with 10 min interval. The experiment results would be excluded if the pressure exceeded 500 g.
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Table 1 Comparisonof glucose concentration among different groups (mM, n = 10, Mean SD). Group
T1
T2
T3
T4
C Mino DM DM + Mino
5.45 0.87 5.28 0.68 5.31 0.72 5.38 1.14
6.33 0.99 5.63 0.89 19.41 3.27* 20.04 2.97*
5.76 0.71 4.63 0.92 21.04 2.40* 22.00 3.44*
5.11 0.32 5.37 0.56 23.04 3.43* 21.35 3.53*
The experiment was carried out before treatment (T1), 2 weeks (T2), 4 weeks (T3) and 8 weeks (T4) post treatment. * P < 0.01 vs Group C.
2.5. Detection of notch-1, iba-1, NICD and hes-1 expression in spinal cord by WB 8 weeks post diabetes induction, the lumbar enlargement (L4 L6) of the spinal cord was retrieved from anesthetized rats and snap frozen in liquid nitrogen. The total protein was extracted, quantified, prepared for SDS-PAGE, and detect by WB using indicated antibodies. The optical density of the bands on the film was analyzed by image analysis system under the chemiluminescence environment. The relative expression level of target proteins was compared to internal reference b-actin or Histon-1.
Table 2 Comparison of body weight among different groups (g, n = 10, Mean SD). Group
T1
T2
T3
T4
C Mino DM DM + Mino
198.28 7.85 197.12 10.54 196.76 2.33 203.20 10.14
243.72 14.52 226.08 12.80 183.44 14.66* 204.16 11.81
305.32 16.17 303.18 15.52 175.32 16.99* 199.64 18.29**
351.96 5.92 355.20 7.77 153.10 15.42* 220.10 15.99**
The body weight was measured before treatment (T1), 2 weeks (T2), 4 weeks (T3) and 8 weeks (T4) post treatment. * P < 0.01 vs Group C. ** P < 0.05 vs Group DM.
Table 3 Minocycline administration improved sural nerve conduction velocity (unit: m/s) in diabetic rats. Group
T1
T2
T3
T4
C Mino DM DM + Mino
50.74 5.31 53.83 10.91 52.06 9.26 51.15 14.76
51.31 5.88 48.29 6.39 35.58 8.09* 39.78 7.77*
50.83 10.74 49.59 5.17 25.32 5.24* 41.02 8.68*~
51.39 10.74 51.13 6.58 24.43 4.70* 44.67 3.45~
Note: compared with C group, *P < 0.01; compared with DM group, ~P < 0.05; The experiment was carried out before treatment (T1), 2 weeks (T2), 4 weeks (T3) and 8 weeks (T4) post treatment.
2.6. Minocycline and DAPT administration Two weeks after diabetes induction, minocycline was dissolved in 0.9% saline and administered ip to both Mino group and DM+ Mino group rats at a dose of 40 mg/kg each day for 3 days. The rats in C group and DM group were given the same volume of saline ip as vehicle control. The sural nerve conduction velocity was measured before treatment (T1), 2 weeks (T2), 4 weeks (T3) and 8 weeks (T4) post treatment. The TWL and MWT were measured weekly (W0-W8) during the same period of time, in which W0
refer to the baseline value undertook before treatment. At week 8, the rats were sacrificed and the Notch-1, Iba-1, NICD and Hes-1 expression were detected by WB in the lumbar enlargement of spinal cord (L4 L6). To study the synergistic effect of minocycline and DAPT on the treatment of DNP, another 40 rats were subjected to STZ administration to induce diabetes and divide into four groups (10 rats/group): DM group, DAPT group, Mino group, and DAPT + Mino group. DAPT (1 mg/kg) was administrated by ip 3 times/
Fig. 1. Minocycline administration significant inhibited Notch-1 expression and activity in diabetic rats. A and C: Male adults SD rats were rendered diabetic and treated with 40 mg/kg minocycline for 2 weeks. The expression of Notch-1 receptor, Iba-1 (A), NICD, and Hes-1 (B) in lumber spinal cord were assessed by WB with indicated antibodies 8 weeks post diabetes induction. B and D: Quantification of the protein levels of Notch-1, Iba-1 (B), NICD, and Hes-1 (D) in (A) and (C). * P < 0.05, ** P < 0.01, *** P < 0.001).
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week. Minocycline was given as above mentioned. The TWL and MWT in each rat were measured from before STZ injection (T0) to the next 8 week (W1-W8). The rats were sacrificed at end time point (W8), and the spinal cord of L4-L6 was collected and subject for WB. 2.7. Statistical analysis The WB bands were scanned and quantified by Quantity One Image Analysis System. The data were presented as mean standard deviation (SD), and analyzed by One-way ANOVA (SPSS 17.0 software). P < 0.05 was considered as statistically significant. 3. Results 3.1. Minocycline treatment accelerated sural nerve conduction velocity in diabetic rats via inhibition notch-1 expression and activity The dramatically increase blood glucose concentration (Table. 1) and attenuated body weight (Table. 2) in DM group suggested that the SD rats were rendered diabetic post 65 mg/kg STZ ip. The sural nerve conduction velocity of diabetic rats was significantly decreased 2 weeks post STZ injection (T2) and continued
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decreasing till 8 weeks (T4) compared to non-diabetic rats (C group) (Table. 3), which is in line with our previously published data[27]. Administration of minocycline in diabetic rats significantly accelerated the attenuated nerve conduction compare with saline vehicle controls (DM group) (Table. 3), suggesting that minocycline is effective in improving the conduction of sciatic nerve in diabetes rats. Consistent with these results, the expression of Notch-1 receptor as well as the receptor activation product NICD and the downstream target Hes-1 was significantly augmented in the luminal spinal cord of STZ-induced diabetic rats (Fig. 1A and D). The expression of macrophage/microglia marker Iba-1 was also upregulated in diabetic mice (Fig. 1A and B), indicating the presence of inflammation and microglia proliferation. Administration of minocycline, however, dramatically blocked Notch-1 expression and activation (Fig. 1 and 2), as well as the expression of Iba-1. 3.2. Administration of minocycline attenuated TWL and MWT in diabetic rats through notch inhibition The threshold for thermal and mechanical hyperalgesia presented as TWL and MWT were gradually decreased in STZinduced diabetic rats (Fig. 2A and B). Chronic administration of
Fig. 2. The combination of Minocycline with DAPT significant alleviated DNP via inhibition the expression and activity of Notch-1. A and B: Male adult SD rats were subjected to STZ to induce diabetes. 40 mg/kg minocycline or DAPT (1 mg/kg) was given alone or in combination. TWL and MWT were recorded weekly for 8 weeks post STZ injection. Mean SD; n = 10/group. Compared with DM group, * p < 0.05, ** p < 0.01.C and E: 8 weeks after STZ induction, the lumber spinal cord (L4-L6) was retrieved and subjected to WB using indicated antibodies. D and F: Quantification of the protein expression of Notch-1, Iba-1 (D), NICD, and Hes-1 (F) in (C) and (E). * P < 0.05, ** P < 0.01.
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Notch inhibitor DAPT or minocycline for 2 weeks markedly prevented the development of thermal and mechanical hyperalgesia (Fig. 2A and B), and inhibited the expression and activity of Notch-1that were exhibited as significantly attenuated expression of Notch-1, NICD, and Hes-1 (Fig. 1, Fig. 2C and F). The expression of Iba-1 was also downregulated (Fig. 2C), indicating that minocycline suppressed Notch-1-dependent inflammation and microglia proliferation in spinal cord. Furthermore, the combination of minocycline and DAPT synergistically improved TWL and MWT (Fig. 2A and B) and inhibited Notch-1 expression and activity (Fig. 2C and F). Thus our studies unrevealed a novel mechanism of minocycline in treatment DNP via blocking Notch signaling and suggested the new regimen by combination of minocycline and DAPT holds big promise in the care and management of diabetes and its complications. 4. Discussion Here, we have identified an important role of Notch-1 in microglia in mediating the development of diabetic neuropathy. Inhibition of Notch-1expression and activity by DAPT and/or minocycline inhibited the microglia proliferation and alleviated thermal and mechanical hyperalgesia that frequently manifested in DNP. Activation of Notch-1-mediated Notch signaling pathway depends on the interaction of Notch-1-expressing cells with neighboring cells that express Notch-1 activators such as Jagged or Delta [28]. Ligand binding to Notch-1 induces the proteolytic release by the c-secretase of the Notch intracellular domain (NICD) that trans-locates to the nucleus to regulate the expression of specific target genes such as HES-1 and HEY-1 [28–30]. We have observed that not only the expression of Notch-1, but also the NICD of Notch-1 receptor as well as the downstream Notch target Hes-1 was augmented post STZ administration, indicating that Notch activity was enhanced in diabetic neuropathy. Our discovery is consistent with the previous finding that Notch signaling is activated during nerve injury [12,16], and inactivation of Notch signal transduction pathway before or after the appearance of pain sensitivity either delayed or prevented the progress of injury-induced neuropathy [16]. The biological effect of Notch activation is cellular context- and tissue environment-dependent. Notch-1 has been shown to play a fundamental role in regulating the fate of neural progenitor’s differentiation and survival in embryonic CNS [31,32]. However, recent findings also indicate that Notch-1 signal guides the commitment of neural progenitors to the glial cell fate [15,33,34]. Giving the fact that microglia and other non-neuron cells plays key roles in sensing neuropathic pain[35,36], it is reasonable to speculate that Notch signaling in microglia might have participated the initiation, development, or the progress of DNP. Thus the focus of our study was to explore Notch function in microglia using the specific microglia inhibitor minocycline. The underlying mechanisms of minocycline-mediated suppression of neuropathic pain include attenuating pro-inflammatory cytokines release and alleviating oxidative and nitrosative stress [35,37], and preventing impaired glial glutamate uptake in the spinal sensory synapses [38], et al. We demonstrated in this study that minocycline ameliorated DNP via down-regulation Notch expression and inactivation of Notch signaling in microglia within spinal cord. In addition, administration of minocycline with Notch inhibitor DAPT further strengthened the therapeutic effect of BAPT in treat diabetic neuropathy. Thus our study discovered an important role of Notch in microglia and suggested this novel regimen has the potential in treating DNP.
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