Intratumoral injection of norcantharidin liposome emulsion hybrid delivery system amplifies the cancer-fighting effects of oral sorafenib against hepatocellular carcinoma.

Interventional therapies are increasingly used in clinical trials for hepatocellular carcinoma (HCC). Sorafenib is the front-line remedy for HCC, however, chemoresistance occurs immutably and affects the effectiveness of treatment. In a previous study, a norcantharidin liposome emulsion hybrid (NLEH) delivery system for HCC was developed. This study aims to examine the therapeutic effects of the combination of intratumoral injection of NLEH and sorafenib in treating HCC. Sorafenib combined with NLEH activated the apoptosis pathway by synergistically upregulating caspase-9, promoting cytotoxicity, apoptosis (64.57%), and G2/M cell cycle arrest (48.96%). Norcantharidin could alleviate sorafenib resistance by counteracting sorafenib-induced phosphorylation of Akt. Additionally, intratumoral injection of NLEH exhibited a sustained accumulation in the tumor within 24 h and didn't distribute to other major organs. Intratumoral injection of NLEH in combination with oral sorafenib displayed the most potent tumor growth inhibitory effect (77.91%) in vivo. H&E staining results and the indicators of the renal and liver function tests demonstrated the safety of this combination therapy. Overall, these results showed that intratumoral injection of NLEH in combination with oral sorafenib treatment represented a rational potential therapeutic option for HCC.

MicroRNA-regulated transcriptome analysis identifies four major subtypes with prognostic and therapeutic implications in prostate cancer.

MicroRNA (miRNA) deregulation plays a critical role in the heterogeneous development of prostate cancer (PCa) by tuning mRNA levels. Herein, we aimed to characterize the molecular features of PCa by clustering the miRNA-regulated transcriptome with non-negative matrix factorization. Using 478 PCa samples from The Cancer Genome Atlas, four molecular subtypes (S-I, S-II, S-III, and S-IV) were identified and validated in two merged microarray and RNAseq datasets with 656 and 252 samples, respectively. Interestingly, the four subtypes showed distinct clinical and biological features after comprehensive analyses of clinical features, multiomic profiles, immune infiltration, and drug sensitivity. S-I is basal/stem/mesenchymal-like and immune-excluded with marked transforming growth factor ß, epithelial-mesenchymal transition and hypoxia signals, increased sensitivity to olaparib, and intermediate prognosis. S-II is luminal/metabolism-active and responsive to androgen deprivation therapy with frequent TMPRSS2-ERG fusion and a good prognosis. S-III is characterized by moderate proliferative and metabolic activity, sensitivity to taxane-based chemotherapy, and intermediate prognosis. S-IV is highly proliferative with moderate EMT and stemness, frequent deletions of TP53, PTEN and RB, and the poorest prognosis; it is also immune-inflamed and sensitive to anti-PD-L1 therapy. Overall, based on miRNA-regulated gene profiles, this study identified four distinct PCa subtypes that could improve risk stratification at diagnosis and provide therapeutic guidance.

Neurotensin Attenuates Nociception by Facilitating Inhibitory Synaptic Transmission in the Mouse Spinal Cord.

Neurotensin (NT) is an endogenous tridecapeptide in the central nervous system. NT-containing neurons and NT receptors are widely distributed in the spinal dorsal horn (SDH), indicating their possible modulatory roles in nociception processing. However, the exact distribution and function of NT, as well as NT receptors (NTRs) expression in the SDH, have not been well documented. Among the four NTR subtypes, NTR2 is predominantly involved in central analgesia according to previous reports. However, the expression and function of NTR2 in the SDH has not yet been directly elucidated. Specifically, it remains unclear how NT-NTR2 interactions contribute to NT-mediated analgesia. In the present study, by using immunofluorescent histochemical staining and immunohistochemical staining with in situ hybridization histochemical staining, we found that dense NT- immunoreactivity (NT-ir) and moderate NTR2-ir neuronal cell bodies and fibers were localized throughout the superficial laminae (laminae I-II) of the SDH at the light microscopic level. In addition, γ-aminobutyric acid (GABA) and NTR2 mRNA were colocalized in some neuronal cell bodies, predominantly in lamina II. Using confocal and electron microscopy, we also observed that NT-ir terminals made both close contacts and asymmetrical synapses with the local GABA-ir neurons. Second, electrophysiological recordings showed that NT facilitated inhibitory synaptic transmission but not glutamatergic excitatory synaptic transmission. Inactivation of NTR2 abolished the NT actions on both GABAergic and glycinergic synaptic release. Moreover, a behavioral study revealed that intrathecal injection of NT attenuated thermal pain, mechanical pain, and formalin induced acute inflammatory pain primarily by activating NTR2. Taken together, the present results provide direct evidence that NT-containing terminals and fibers, as well as NTR2-expressing neurons are widely distributed in the spinal dorsal horn, GABA-containing neurons express NTR2 mainly in lamina II, GABA coexists with NTR2 mainly in lamina II, and NT may directly increase the activity of local inhibitory neurons through NTR2 and induce analgesic effects.

Systemic Injection of Thalidomide Prevent and Attenuate Neuropathic Pain and Alleviate Neuroinflammatory Response in the Spinal Dorsal Horn.

BACKGROUND AND OBJECTIVE: Thalidomide (Tha) has been shown to exert immunomodulatory and anti-inflammatory properties. Whether Tha can alleviate spinal nerve ligation (SNL)-induced neuropathic pain (NP) is still unclear. This study aimed to investigate the therapeutic effect of Tha on the SNL-induced NP and further explore the potential analgesic mechanisms of Tha. METHODS: The effects of Tha on SNL-induced mechanical allodynia were assessed by pain behavioral testing. The expressions of the astrocyte marker glial fibrillary acidic protein (GFAP) and the microglia marker Iba1 in the spinal dorsal horn were evaluated by immunofluorescence histochemistry. Protein expressions of the tumor necrosis factor alpha (TNF-α) in the spinal dorsal horn were tested by Western blot assay. Data were analyzed using one-way ANOVA or two-way ANOVA. RESULTS: By the pretreatment with a single intraperitoneal injection, the PWMT in SNL+Tha group was significantly increased from day 1 to day 2 after SNL (P < 0.05 compared with SNL+Veh group). By the posttreatment with a single intraperitoneal injection, the PWMT in SNL+Tha group was also significantly increased from day 3 to day 4 after SNL (P < 0.05 compared with SNL+Veh group). By the posttreatment with multiple intraperitoneal injection, both the PWMT and the PWTL in SNL+Tha group were similarly significantly increased from day 3 to day 14 after SNL (P < 0.05 compared with SNL+Veh group). Furthermore, the GFAP and Iba1 expressions and TNF-α levels of the ipsilateral spinal dorsal horn in SNL+Tha group were significantly weaker from day 3 to day 14 after SNL than those in SNL+Veh group (P < 0.05). CONCLUSION: Tha can significantly alleviate NP induced by SNL. The analgesic mechanism may be related to inhibition of astrocyte and microglia activation as well as down-regulation of TNF-α levels in the spinal dorsal horn.

Vesicular glutamate transporter isoforms: The essential players in the somatosensory systems.

In nervous system, glutamate transmission is crucial for centripetal conveyance and cortical perception of sensory signals of different modalities, which necessitates vesicular glutamate transporters 1-3 (VGLUT 1-3), the three homologous membrane-bound protein isoforms, to load glutamate into the presysnaptic vesicles. These VGLUTs, especially VGLUT1 and VGLUT2, selectively label and define functionally distinct neuronal subpopulations at each relay level of the neural hierarchies comprising spinal and trigeminal sensory systems. In this review, by scrutinizing each structure of the organism's fundamental hierarchies including dorsal root/trigeminal ganglia, spinal dorsal horn/trigeminal sensory nuclear complex, somatosensory thalamic nuclei and primary somatosensory cortex, we summarize and characterize in detail within each relay the neuronal clusters expressing distinct VGLUT protein/transcript isoforms, with respect to their regional distribution features (complementary distribution in some structures), axonal terminations/peripheral innervations and physiological functions. Equally important, the distribution pattern and characteristics of VGLUT1/VGLUT2 axon terminals within these structures are also epitomized. Finally, the correlation of a particular VGLUT isoform and its physiological role, disclosed thus far largely via studying the peripheral receptors, is generalized by referring to reports on global and conditioned VGLUT-knockout mice. Also, researches on VGLUTs relating to future direction are tentatively proposed, such as unveiling the elusive differences between distinct VGLUTs in mechanism and/or pharmacokinetics at ionic/molecular level, and developing VGLUT-based pain killers.

The behavioral study on the interactive aggravation between pruritus and depression.

BACKGROUND: The interactive aggravation of pruritus and depression is well-known, but an appropriate experimental model that could mimic this behavioral phenomenon is still lacking. Thus, a systematic animal behavioral investigation was carried out in this study. This will promote the research and treatment of pruritus and depression. METHODS: The 2,4-dinitrofluorobenzene (DNFB)-induced chronic itch model was established to measure the depression index by forced swimming test (FST), tail suspension test (TST), and splash test (ST). The chronic unpredicted mild stress (CUMS)-induced depression model was established to measure spontaneous itch and acute histamine or chloroquine-induced itch behaviors. A depression and itch combining model was also established to measure the scratching and depression behaviors. The motor function of DNFB mice was analyzed by the rotarod test. RESULTS: The scratching number, the immobility time in the FST and TST, and the grooming number in the ST test were all significantly increased in the chronic itch model. Mice receiving CUMS treatment showed significantly increased spontaneous scratching number, immobility time in the FST and TST tests, and grooming number in the ST. The combined model showed increased immobility time in FST and TST tests and increased grooming number in ST comparing to the depression model, and showed increased scratching number comparing to the chronic itch model. After histamine (His) or chloroquine (CQ) injection, the scratching numbers of CUMS mice were all significantly increased compared to those of His- and CQ-control, respectively. Anti-depression drug ketamine could significantly inhibit the depression-like behaviors of CUMS mice, and simultaneously stopped the promoting effect on His-induced acute itch. CONCLUSIONS: This study established an appropriate cross aggravation experimental mode and demonstrated that there is cross aggravation between pruritus and depression. The illumination of related mechanisms underlying this cross aggravation effect will provide theoretical basis for the prevention and treatment of depression and pruritus.

[Effects of different straw recycling and tillage methods on soil respiration and microbial activity].

To explore the effects of different tillage methods and straw recycling on soil respiration and microbial activity in summer maize field during the winter wheat and summer maize double cropping system, substrate induced respiration method and CO2 release method were used to determine soil microbial biomass carbon, microbial activity, soil respiration, and microbial respiratory quotient. The experiment included 3 tillage methods during the winter wheat growing season, i.e., no-tillage, subsoiling and conventional tillage. Each tillage method was companied with 2 straw management patterns, i.e., straw recycling and no straw. The results indicated that the conservation tillage methods and straw recycling mainly affected 0-10 cm soil layer. Straw recycling could significantly improve the microbial biomass carbon and microbial activity, while decrease microbial respiratory quotient. Straw recycling could improve the soil respiration at both seedling stage and anthesis, however, it could reduce the soil respiration at filling stage, wax ripeness, and harvest stage. Under the same straw application, compared with conventional tillage, the soil respiration and microbial respiratory quotient in both subsoiling and no-tillage were reduced, while the microbial biomass carbon and microbial activity were increased. During the summer maize growing season, soil microbial biomass carbon and microbial activity were increased in straw returning with conservation tillage, while the respiratory quotient was reduced. In 0-10 cm soil layer, compared with conventional tillage, straw recycling with subsoiling and no-tillage significantly increased soil microbial biomass carbon by 95.8% and 74.3%, and increased soil microbial activity by 97.1% and 74.2%, respectively.

The roles of neurotensin and its analogues in pain.

Neurotensin (NT) is an endogenous 13 amino acid neuropeptide with profound opioid-independent analgesic effects. This role of NT is thought to be mediated by both neurotensin receptor subtype 1 (NTS1) and neurotensin receptor subtype 2 (NTS2). NT and its receptors are widely distributed in the pain circuits in central nervous system. Thus NT might modulate pain in many structures of pain pathway, such as spinal cord, rostroventral medulla (RVM) and periaqueductal gray (PAG). Actually either intrathecal application of NT or direct injection of NT into RVM or PAG or intracerebroventricular injection of NT showed analgesic effects. NT exerted its antinociceptive effects in both acute pain and chronic pain models. The analgesic effects of NT were originally found in acute pain experiments. In the case of pathological pain, for example, formalin injection induced inflammatory pain and sciatic nerve constriction induced neuropathic pain, NT also shows antinociceptive effects. The effects exist in somatic pain as well as visceral pain induced by noxious colorectal distension (CRD) or writhing test. It should be noted that NT plays an important role in stress-induced antinociception (SIAN), especially in higher intensity stress experiments. However as a neuropeptide, NT is susceptible to degradation by peptidases and cannot cross the blood-brain barrier (BBB). Great efforts have been made to find NT analogues that are more biologically stable and could inhibit pain by systematic administration. The present review focuses on the analgesic role and the underlying mechanisms of NT and its analogues in pain, especially in chronic pain models.

Morphological evidence for a neurotensinergic periaqueductal gray-rostral ventromedial medulla-spinal dorsal horn descending pathway in rat.

Neurotensin (NT) is an endogenous neuropeptide that exerts potent opioid-independent analgesic effects, most likely via the type 2 NT receptor (NTR2). Previous morphological and electrophysiological studies suggested that the NT-NTR2 system is primarily localized in structures that constitute the descending pain control pathway, such as the periaqueductal gray (PAG), the rostral ventromedial medulla (RVM), and the spinal dorsal horn (SDH). However, relevant morphological evidence for this neurotensinergic (NTergic) circuit is lacking. Thus, the aim of the present study was to morphologically elucidate the potential sites and connections in the NT-NTR2 system that are involved in the descending pain control pathway. Based on light and electron microscopy combined with anterograde and retrograde tracing, we found evidence that NTR2-immunoreactive (IR) neurons in the RVM receive NT-IR projections originating from the PAG; express NT, serotonin (5-HT), or both; and send projections that terminate in laminae I and II of the SDH. These results suggest that NTR2 may contribute to pain control by binding to NT in the PAG-RVM-SDH pathway. In conclusion, our data provide morphological evidence for an NTergic PAG-RVM-SDH pathway, implicating novel mechanisms of NT-induced analgesia.

Development and applications of a nasopharyngeal carcinoma Tet-Off cell line.

The conditional activation and inactivation of target gene expression in a nasopharyngeal carcinoma (NPC) cell line is beneficial for the study of the roles of NPC-related genes. Based on the Tet-Off Advanced system, a NPC S18 Tet-Off cell line was developed by stable transfection of a pTet-Off Advanced vector (regulator plasmid in Tet-Off Advanced system) into NPC S18 cells. Doxycycline-dependent regulators expressed in the S18 Tet-Off cells were examined by transient and stable transfection of pTRE-Tight-Luc. The S18 Tet-Off-Luc clone selected by stable transfection of pTRE-Tight-Luc into S18 Tet-Off cells expressed firefly luciferase under tight control of doxycycline in a time- and dose-dependent manner. To test applications of the S18 Tet-Off cell line in the study of gene function, the impact of ferritin heavy chain (FTH1) gene on NPC cell growth was examined. The S18 Tet-Off-FTH1 clone was developed by stably transfecting pTRE-Tight-FTH1 (response plasmid harboring FTH1) into S18 Tet-Off cells. FTH1 levels in the S18 Tet-Off-FTH1 clone were semi-quantitatively regulated in response to varying concentrations of doxycycline. A cell proliferation assay showed that a high expression of FTH1 (cells grown in the absence of doxycycline) reduced cell growth, while moderate FTH1 overexpression (cells grown in 0.1 ng/ml doxycycline) had no adverse effect on cell growth. In conclusion, the S18 Tet-Off cell line provides a proven genetic background for convenient access to controllable gene expression in NPC.

The synaptic connectivity that underlies the noxious transmission and modulation within the superficial dorsal horn of the spinal cord.

Noxious stimuli can usually cause the aversive sensations, pain and itch. The initial integration of such noxious information occurs in the superficial dorsal horn of the spinal cord (SDH), which is very important for understanding pain sensation and developing effective analgesic strategies. The circuits formed by pools of neurons and terminals within SDH are accepted as the platform for such complicated integrations and are highly plastic under conditions of inflammatory or neuropathic pain. Recent literature offers a complicated, yet versatile view of SDH intrinsic circuits with both inhibitory and excitatory components. However, our knowledge about the adaptative regulation of SDH local circuits is still far from sufficient due to the incomplete understanding of their organization as they are intermingled with primary afferent fibers (PAFs), poorly understood or identified SDH neurons, somehow contradictory data for descending control systems. A more positive view emphasizes abundant modern data on SDH neuron morphology and physiology riding on the back of significant technological advancements used in neuroscience. Reviewing the current literature on this topic thus produced an integrated understanding of SDH neurons and the SDH local circuits involved in noxious transmission and modulation.

Endomorphin-1 is more potent than endomorphin-2 in inhibition of synaptic transmission in substantia gelatinosa of adult rat spinal cord.

Effects of endomorphin-1 (EM-1) and endomorphin-2 (EM-2) on synaptic trans-mission were investigated on neurons in substantia gelatinosa (SG) of the spinal dorsal horn. Both EM-1 (1 microM) and EM-2 (1 microM) remarkably reduced the frequency but not the amplitude of miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs). These effects were antagonized by beta-funaltrexamine (beta-FNA, 10 microM), a selective mu-opioid receptor antagonist. Noticeably, EM-1 showed higher potency in decreasing the frequency of mEPSCs and mIPSCs than that of EM-2. These results indicate that EMs suppress both excitatory and inhibitory synaptic transmission by activating presynaptic mu-opioid receptors in the SG and EM-1, compared with EM-2, might be a more potent endogenous analgesic at the spinal cord level.

Morphological evidence for GABA/glycine-cocontaining terminals in synaptic contact with neurokinin-1 receptor-expressing neurons in the sacral dorsal commissural nucleus of the rat.

Previous studies have shown that neurons in the sacral dorsal commissural nucleus (SDCN) express neurokinin-1 receptor (NK1R) and can be modulated by the co-release of GABA and glycine (Gly) from single presynaptic terminal. These results raise the possibility that GABA/Gly-cocontaining terminals might make synaptic contacts with NK1R-expressing neurons in the SDCN. In order to provide morphological evidence for this hypothesis, the triple-immunohistochemical studies were performed in the SDCN. Triple-immunofluorescence histochemical study showed that some axon terminals in close association with NK1R-immunopositive (NK1R-ip) neurons in the SDCN were immunopositive for both glutamic acid decarboxylase (GAD) and glycine transporter 2 (GlyT2). In electron microscopic dual- and triple-immunohistochemistry for GAD/GlyT2, GAD/NK1R, GlyT2/NK1R, or GAD/GlyT2/NK1R also revealed dually labeled (GAD/GlyT2-ip) synaptic terminals upon SDCN neurons, as well as GAD- and/or GlyT2-ip axon terminals in synaptic contact with NK1R-ip SDCN neurons. These results suggested that some synaptic terminals upon NK1R-expressing SDCN neurons co-released both GABA and Gly.

Vesicular glutamate transporter immunoreactivity in the central and peripheral endings of muscle-spindle afferents.

Expression of vesicular glutamate transporters (VGLUTs: VGLUT1, VGLUT2 and VGLUT3) in muscle spindle afferents was examined in rats. VGLUT1 immunoreactivity was detected in the sensory endings on the equatorial and juxta-equatarial regions of intrafusal fibers as well as in many axon terminals within lamina IX of the spinal cord. VGLUT1 might be expressed not only in the central axon terminals but also in the peripheral sensory endings of muscle-spindle afferents.

Origins of GABA(B) receptor-like immunoreactive terminals in the rat spinal dorsal horn.

By means of immunohistochemistry for gamma-aminobutyric acid receptor B subtype (GABA(B)R), the origins of GABA(B)R-like immunoreactive (GABA(B)R-LI) terminals in the rat spinal dorsal horn were investigated. After dorsal root rhizotomy and/or spinal cord hemisection, the densities of GABA(B)R-LI terminals were remarkably depleted in the ipsilateral superficial dorsal horn of relevant segments, whereas GABA(B)R-LI neurons and sparsely distributed GABA(B)R-LI terminals remained. After injection of Fluoro-Gold (FG) into the left side of superficial lumbar dorsal horn, FG retrograde-labeled neurons were mainly observed in the ipsilateral rostral ventromedial medulla (RVM) and brainstem raphe nuclei. Some of the FG-labeled neurons, especially in the RVM, exhibited GABA(B)R-like immunoreactivity. Additionally, immunofluorescence histochemical double-staining revealed that the majority of GABA(B)R-LI neurons in the periaqueductal gray (PAG), RVM and brainstem raphe nuclei showed 5-hydroxytryptamine (5-HT)-like immunoreactivity. The present study morphologically proves that GABA(B)R-LI terminals in the spinal dorsal horn originate from peripheral afferents, intrinsic neurons and supraspinal structures; GABA(B)R and 5-HT co-exist in many neurons in the PAG, RVM and brainstem raphe nuclei. Considering that PAG, RVM, brainstem raphe nuclei and spinal dorsal horn are important structures involved in the pain modulation, we suggest that the descending pain modulation system might be mediated, at least in part, by GABA(B)R.

Activation of capsaicin receptors on the sciatic nerve induces FOS expression in the spinal dorsal horn of adult rats.

By using immunohistochemical staining for FOS protein in the spinal cord, the role of capsaicin receptors on the sciatic nerve was investigated. After topical application of capsaicin (1%) to the sciatic nerve, FOS-like immunoreactive (FOS-LI) neurons were observed, chiefly in the superficial laminae of the lumbar dorsal horn. Topical application of capsazepine (5%) or lidocaine (2%) to the sciatic nerve for 15 min before the application of capsaicin reduced the number of FOS-LI neurons in the superficial dorsal horn (by 83.2 +/- 1.7 and 32.4 +/- 1.2%, respectively). One week after pretreatment of the sciatic nerve with colchicine, the number of FOS-LI neurons induced by capsaicin was greatly decreased (by 74.6 +/- 1.7%). Given that FOS protein expression after peripheral noxious stimulation is found in a location similar to that in the present study, our results indicate that the capsaicin receptor on the sciatic nerve is involved in the transmission of noxious information.

Norepinephrine depresses the capsaicin-evoked miniature excitatory postsynaptic currents in substantia gelatinosa of the rat spinal cord.

Capsaicin selectively excites nociceptive primary afferent fibers and increases the frequency of glutaminergic miniature excitatory postsynaptic currents (mEPSCs) in the substantia gelatinosa of the spinal dorsal horn. The whole-cell voltage-clamp recording technique was used to examine the effect of norepinephrine (NE) on the capsaicin-induced increase in the frequency of mEPSCs. In the presence of tetrodotoxin, bath application of capsaicin (1 microM) remarkably enhanced the frequency of mEPSCs (295+/-52% of control). Following pretreatment with NE (10 microM), the capsaicin-induced frequency facilitation of mEPSCs was significantly depressed to 151+/-17% of the control. NE-induced depression in capsaicin action was blocked by yohimbine, a selective alpha(2)-adrenergic receptor antagonist, indicating that NE exerts depression by activating the alpha(2)-adrenergic receptor. As the postsynaptic action of NE has been precluded in the present study, the results suggest that NE inhibits nociceptive input at a presynaptic site, the primary afferent terminal, during the nociceptive transmission in the spinal dorsal horn.