Effects of molecular weight fraction on antioxidation capacity of rice protein hydrolysates.

Rice protein was used as a starting material to provide rice protein hydrolysates (RPH) through enzyme-assisted extraction. RPH was further fractionated using ultrafiltration membrane (UF) and classified by molecular weight (MW; MW < 1 kDa, MW 1-10 kDa, and MW > 10 kDa). Peptides with MW < 1 kDa possessed superior antioxidant properties (p < 0.05). Therefore, UF demonstrated great efficacy in selectively separating antioxidant peptides. A Pearson correlation analysis revealed that the total phenolic concentration was correlated with oxygen radical absorbance capacity (ORAC; r = 0.999, p < 0.05). Amino acid contents had negative correlations with the scavenging activity (specifically, IC50) of 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radicals (r = - 0.986 to - 1.000). Reducing power was related to aromatic amino acid contents (r = 0.997, p < 0.05). In this study, enzymatic hydrolysis was discovered to be an effective method of extracting and isolating natural antioxidant proteins from broken rice, thus preserving the nutritional quality of rice and making those proteins more accessible in future applications.

Evaluating the Antioxidants, Whitening and Antiaging Properties of Rice Protein Hydrolysates.

Plant-derived protein hydrolysates have potential applications in nutrition. Rice protein hydrolysates (RPHs), an excellent source of proteins, have attracted attention for the development of cosmeceuticals. However, few studies have reported the potential application of RPH in analysis, and this study examined their antioxidant activities and the inhibitory activities of skin aging enzymes. The results indicated that the total phenolic and flavonoid concentrations were 2.06 ± 0.13 mg gallic acid equivalent/g RPHs and 25.96 ± 0.52 µg quercetin equivalent/g RPHs, respectively. RPHs demonstrated dose-dependent activity for scavenging free radicals from 1,1-diphenyl-2-picrylhydrazyl [half-maximal inhibitory concentration (IC50) = 42.58 ± 2.1 mg/g RPHs] and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (IC50 = 2.11 ± 0.88 mg/g RPHs), dose-dependent reduction capacity (6.95 ± 1.40 mg vitamin C equivalent/g RPHs) and oxygen radical absorbance capacity (473 µmol Trolox equivalent/g RPHs). The concentrations of the RPH solution required to achieve 50% inhibition of hyaluronidase and tyrosinase activities were determined to be 8.91 and 107.6 mg/mL, respectively. This study demonstrated that RPHs have antioxidant, antihyaluronidase, and antityrosinase activities for future cosmetic applications.

An in vivo mouse model of long-term potentiation at synapses between primary afferent C-fibers and spinal dorsal horn neurons: essential role of EphB1 receptor.

BACKGROUND: Long-term potentiation (LTP), a much studied cellular model of synaptic plasticity, has not been demonstrated at synapses between primary afferent C-fibers and spinal dorsal horn (DH) neurons in mice in vivo. EphrinB-EphB receptor signaling plays important roles in synaptic connection and plasticity in the nervous system, but its role in spinal synaptic plasticity remains unclear. RESULTS: This study characterizes properties of LTP at synapses of C-fibers onto neurons in the superficial DH following high-frequency stimulation (HFS) of a peripheral nerve at an intensity that activates C-fibers and examines associated activation of Ca2+/calmodulin-activated protein kinase II (p-CaMKII), extracellular signal-regulated kinase (p-ERK) and the cyclic AMP response element binding protein (p-CREB) and expression of c-Fos, and it investigates further roles for the EphB1 receptor in LTP. HFS induced LTP within 5 min and lasts for 3-8 h during the period of recording and resulted in upregulation of p-CaMKII, p-ERK and p-CREB protein levels in the spinal cord and expression of c-Fos in DH. Intrathecal pretreatment of MK-801 or EphB2-Fc prevented LTP and significantly reduced upregulation of p-CaMKII, p-ERK, p-CREB and c-Fos. Further, targeted mutation of EphB1 receptor prevented induction of LTP and associated increases in phosphorylation of CaMKII, ERK, and CREB. CONCLUSION: This study provides an in vivo mouse model of LTP at synapses of C-fibers onto the superficial DH neurons that will be valuable for studying the DH neuron excitability and their synaptic plasticity and hyperalgesia. It further takes advantage of examining functional implications of a specific gene targeted mice and demonstrates that the EphB1 receptor is essential for development of LTP.

EphrinB-EphB receptor signaling contributes to neuropathic pain by regulating neural excitability and spinal synaptic plasticity in rats.

Bidirectional signaling between ephrins and Eph receptor tyrosine kinases was first found to play important roles during development, but recently has been implicated in synaptic plasticity and pain processing in the matured nervous system. We show that ephrinB-EphB receptor signaling plays a critical role is induction and maintenance of neuropathic pain by regulating neural excitability and synaptic plasticity in the dorsal root ganglion (DRG) and the spinal dorsal horn (DH). Intrathecal application of blocking reagents for EphB-receptors, EphB1-Fc and EphB2-Fc chimeras inhibits the induction and maintenance of nerve injury-induced thermal hyperalgesia and mechanical allodynia. These blockers also prevent and suppress the nerve injury-induced hyperexcitability of nociceptive small DRG neurons, sensitization of DH neurons and long-term potentiation (LTP) of synapses between C fibers and DH neurons. In naïve, uninjured animals intrathecal administration of EphB-receptor activators ephrinB1-Fc and ephrinB2-Fc, respectively, induces thermal hypersensitivity and lowers the threshold for LTP, while EphB1-Fc prevents induction of the LTP. Western Blot analysis shows that nerve injury triggers an upregulation of the ephrinB1 and EphB1 receptor proteins in DRG and the spinal cord. These results indicate that, by regulating excitability of nociceptive-related neurons in DRG and DH and the synaptic plasticity at the spinal level, ephrinB-EphB receptor signaling contributes to neuropathic pain. This novel role for ephrinB-EphB receptor signaling suggests that these molecules may be useful therapeutic targets for treating pain after nerve injury.

Upregulation and redistribution of ephrinB and EphB receptor in dorsal root ganglion and spinal dorsal horn neurons after peripheral nerve injury and dorsal rhizotomy.

EphrinB-EphB receptor signaling plays diverse roles during development, but recently has been implicated in synaptic plasticity in the matured nervous system and in pain processes. The present study investigated the correlation between expression of ephrinB and EphB receptor proteins and chronic constriction injury (CCI) of the sciatic nerve and dorsal rhizotomy (DR) in dorsal root ganglion (DRG) and spinal cord (SC); and interaction of CCI and DR on expression of these signals. Adult, male Sprague-Dawley rats were employed and thermal sensitivity was determined in the sham operated CCI and DR rats. Western blot and immunobiochemistry analysis and immunofluorescence staining techniques were used to detect the expression and location of the ephrinB-EphB receptor proteins in DRG and SC. The results showed that expression of ephrinB1 and EphB1 receptor proteins was significantly upregulated in DRG and SC in a time-dependent manner corresponding to the development of thermal hyperalgesia after CCI. The increased expression is predominately located in the medium- and small-sized DRG neurons, the superficial layers of spinal dorsal horn (DH) neurons, and the IB4 positive nociceptive terminals. DR increases ephrinB1 in DRG, not SC and EphB receptor in SC, not DRG. DR suppressed CCI-induced upregulation of ephrinB1 in SC and EphB1 receptor in DRG and SC. These findings indicate that ephrinB-EphB receptor activation and redistribution in DRG and DH neurons after nerve injury could contribute to neuropathic pain. This study may also provide a new mechanism underlying DR-induced analgesia in clinic.

Dissociation of dorsal root ganglion neurons induces hyperexcitability that is maintained by increased responsiveness to cAMP and cGMP.

Injury or inflammation affecting sensory neurons in dorsal root ganglia (DRG) causes hyperexcitability of DRG neurons that can lead to spontaneous firing and neuropathic pain. Recent results indicate that after chronic compression of DRG (CCD treatment), both hyperexcitability of neurons in intact DRG and behaviorally expressed hyperalgesia are maintained by concurrent activity in cAMP-protein kinase A (PKA) and cGMP-protein kinase G (PKG) signaling pathways. We report here that when tested under identical conditions, dissociation produces a pattern of hyperexcitability in small DRG neurons similar to that produced by CCD treatment, manifest as decreased action potential (AP) current threshold, increased AP duration, increased repetitive firing to depolarizing pulses, increased spontaneous firing and resting depolarization. A novel feature of this hyperexcitability is its early expression-as soon as testing can be conducted after dissociation (approximately 2 h). Both forms of injury increase the electrophysiological responsiveness of the neurons to activation of cAMP-PKA and cGMP-PKG pathways as indicated by enhancement of hyperexcitability by agonists of these pathways in dissociated or CCD-treated neurons but not in control neurons. Although inflammatory signals are known to activate cAMP-PKA pathways, dissociation-induced hyperexcitability is unlikely to be triggered by signals released from inflammatory cells recruited to the DRG because of insufficient time for recruitment during the dissociation procedure. Inhibition by specific antagonists indicates that continuing activation of cAMP-PKA and cGMP-PKG pathways is required to maintain hyperexcitability after dissociation. The reduction of hyperexcitability by blockers of adenylyl cyclase and soluble guanylyl cyclase after dissociation suggests a continuing release of autocrine and/or paracrine factors from dissociated neurons and/or satellite cells, which activate both cyclases and help to maintain acute, injury-induced hyperexcitability of DRG neurons.

Abeta-afferents activate neurokinin-1 receptor in dorsal horn neurons after nerve injury.

We provide new evidence demonstrating that peripheral nerve injury produces profound alterations in synaptic input to dorsal horn neurons mediated by non-nociceptive sensory neurons, and activation of neurokinin-1 receptor may be involved in the enhanced synaptic response and thus contribute to the tactile allodynia. Our results show that Abeta-fiber-evoked field potential significantly increased in the first postoperative week and decreased thereafter while maximal mechanical allodynia was exhibited. The neurokinin-1 receptor antagonist L703,606 significantly reduced Abeta-fiber-evoked field potential in nerve-injured but not in sham-operated animals. The non-N-methyl-D-aspartate receptor antagonist CNQX inhibited Abeta-fiber-evoked field potential in both nerve-injured and sham-operated rats, while the N-methyl-D-aspartate receptor antagonist MK-801 did not affect Abeta-fiber-evoked field potential in either CCI or sham-operated animals.

Age-related changes in deterministic behaviors of nociceptive firing of rat dorsal horn neurons.

To demonstrate the age-related changes in the dynamics of the nociceptive discharge of dorsal horn nociceptive neurons, the nonlinear prediction method was used to quantify the degree of deterministic behavior within the interspike interval series of tissue injury-induced firing of spinal nociceptive neurons in anesthetized adult young (3-4 months) and aged (>22 months) rats. Subcutaneous bee venom injection induced long-term discharge of spinal wide dynamic range (WDR) neurons in both groups. However, the nociceptive discharge of single WDR neurons in the aged group showed higher determinism when compared with the adult young rats. This result suggests that the dynamics of single nociceptive neurons may not remain constant throughout the life span, and this age-associated change may be an underlying mechanism for various pain manifestations in the elderly population.

Complexity of tissue injury-induced nociceptive discharge of dorsal horn wide dynamic range neurons in the rat, correlation with the effect of systemic morphine.

Persistent discharge of wide dynamic range (WDR) neurons was recorded from lumbar dorsal horn of anesthetized rats following subcutaneous bee venom injection into the receptive field. To quantitatively describe the complexity of this nociceptive activity, we computed the approximate entropy (ApEn) for each sampled interspike interval (ISI) series. A larger value of ApEn indicates higher complexity or less regularity and vice versa. The ApEn value varied across different WDR neurons tested, and for each neuron the ApEn remained constant through the 1-h discharge though the average ISI of the sampled data increased progressively with time (16 neurons). A low dose of intravenous morphine (0.3 mg/kg) depressed the activity of WDR neurons differentially, and the degree of this inhibition showed a significant correlation with the value of ApEn (P<0.001, 27 neurons, Spearman's correlation test). The present results suggest that the complexity feature of WDR neurons is various under tissue injury state, and for each single WDR neuron the complexity feature is relatively independent of the strength of peripheral noxious input and cannot be fully described in terms of average firing rate. Moreover, the response of the nociceptive discharge to analgesics may be related to the nonlinear dynamics feature of nociceptive neurons, which can be quantitatively characterized by the degree of complexity.

Detection of deterministic behavior within the tissue injury-induced persistent firing of nociceptive neurons in the dorsal horn of the rat spinal cord.

To unravel the temporal features of the peripheral tissue injury induced persistent nociceptive discharge, single wide dynamic range (WDR) unit activity was recorded extracellularly in lumbar dorsal horn of anesthetized rats and interspike interval (ISI) series were obtained. Subcutaneous (s.c.) bee venom (BV) injection induced persistent discharge of spinal WDR neurons and has been well established to be a good model in evaluation of tissue injury induced pain. By applying a more novel approach, i.e., the unstable periodic orbit (UPO) identification method, we detected a family of significant separate UPOs (period-1, 2 and 3 orbits) within the ISI series of BV-induced nociceptive discharge, but not spontaneous background activity of spinal WDR neuron. Furthermore, temporally dynamic changes of UPOs at lower period-1, 2 and 3 for 4 successive time segments within 1 h time course of WDR unit firing showed temporally dynamic changes, i.e., new orbits with longer ISIs emerged and those with shorter ISIs vanished with time change. By using this method we suggest that BV-induced nociceptive discharge of spinal WDR neuron be a kind of deterministic activity and various UPOs may play some role in temporal coding of sensory information.