BDNF/TrkB Signaling Inhibition Suppresses Astrogliosis and Alleviates Mechanical Allodynia in a Partial Crush Injury Model

Neuropathic pain presents a formidable clinical challenge due to its persistent nature and limited responsiveness to conventional analgesic treatments. While significant progress has been made in understanding the role of spinal astrocytes in neuropathic pain, their contribution and functional changes following a partial crush injury (PCI) remain unexplored. In this study, we investigated structural and functional changes in spinal astrocytes during chronic neuropathic pain, employing a partial crush injury model. This model allowes us to replicate the transition from initial nociceptive responses to persistent pain, highlighting the relevance of astrocytes in pain maintenance and sensitization. Through the examination of mechanical allodynia, a painful sensation in response to innocuous stimuli, and the correlation with increased levels of brain-derived neurotrophic factor (BDNF) along with reactive astrocytes, we identified a potential mechanistic link between astrocytic activity and BDNF signaling.Ultimately, our research provides evidence that inhibiting astrocyte activation through a BDNF/TrkB inhibitor alleviates mechanical allodynia, underscoring the therapeutic potential of targeting glial BDNF-related pathways for pain management. These findings offer critical insights into the cellular and molecular dynamics of neuropathic pain, paving the way for innovative and targeted treatment strategies for this challenging condition.

Quisqualis indica extract ameliorates low urinary tract symptoms in testosterone propionate-induced benign prostatic hyperplasia rats / 한국실험동물학회지

Benign prostate hyperplasia (BPH) is a common disease in old-age males, accounting for approximately 77% of morbidity within the age range of 40 to 70 years. It has been shown that morbidity increases with social graying. Quisqualis indica linn (QI) has been used to treat inflammation, stomach pain, and digestion problems. In this study, we evaluated the symptom-regulating effects of QI extract on a testosterone-induced BPH rat model. After inducing BPH in rats using testosterone propionate (TP) injection, we assessed basal intraurethral pressure (IUP) and increments of IUP elicited by electrical field stimulation (5 V, 5, 10, or 20 Hz) or phenylephrine (Phe) (0.01, 0.03, 0.1 mg/kg IV). To induce BPH, 8-week-old rats were subjected to a daily subcutaneous TP (3 mg/kg) injection for 4 weeks. Finasteride (Fina) (10 mg/kg PO) was administered to the rats in the first treatment, while QI (150 mg/kg PO) was administered to those in the second group. Blood pressure was measured together with IUP, after which low urinary tract (LUT), ventral prostate (VP), testicle, and corpus spongiosum were isolated and weighed. Basal IUPs for the Fina- and QI-treated groups were 87.6 and 86.8%, respectively.LUT and VP organ weights in the QI group were lower than those in the Fina group. However, the QI group showed significantly reduced electrical stimulated or Phe-induced IUP increment compared to the Fina and BPH groups. These results proved that QI can be beneficial for BPH symptoms by inhibiting 5α-reductase and consequently decreasing prostate and releasing urinary pressure.

Onion peel extract and its constituent, quercetin inhibits human Slo3 in a pH and calcium dependent manner

Sperm function and male fertility are closely related to pH dependent K⁺ current (KSper) in human sperm, which is most likely composed of Slo3 and its auxiliary subunit leucine-rich repeat-containing protein 52 (LRRC52). Onion peel extract (OPE) and its major active ingredient quercetin are widely used as fertility enhancers; however, the effect of OPE and quercetin on Slo3 has not been elucidated. The purpose of this study is to investigate the effect of quercetin on human Slo3 channels. Human Slo3 and LRRC52 were co-transfected into HEK293 cells and pharmacological properties were studied with the whole cell patch clamp technique. We successfully expressed and measured pH sensitive and calcium insensitive Slo3 currents in HEK293 cells. We found that OPE and its key ingredient quercetin inhibit Slo3 currents. Inhibition by quercetin is dose dependent and this degree of inhibition decreases with elevating internal alkalization and internal free calcium concentrations. Functional moieties in the quercetin polyphenolic ring govern the degree of inhibition of Slo3 by quercetin, and the composition of such functional moieties are sensitive to the pH of the medium. These results suggest that quercetin inhibits Slo3 in a pH and calcium dependent manner. Therefore, we surmise that quercetin induced depolarization in spermatozoa may enhance the voltage gated proton channel (Hv1), and activate non-selective cation channels of sperm (CatSper) dependent calcium influx to trigger sperm capacitation and acrosome reaction.

Functional identification of protein phosphatase 1-binding consensus residues in NBCe1-B

Protein phosphatase 1 (PP1) is involved in various signal transduction mechanisms as an extensive regulator. The PP1 catalytic subunit (PP1c) recognizes and binds to PP1-binding consensus residues (FxxR/KxR/K) in NBCe1-B. Consequently, we focused on identifying the function of the PP1-binding consensus residue, ⁹²²FMDRLK⁹²⁷ , in NBCe1-B. Using site-directed mutagenesis and co-immunoprecipitation assays, we revealed that in cases where the residues were substituted (F922A, R925A, and K927A) or deleted (deletion of amino acids 922–927), NBCe1-B mutants inhibited PP1 binding to NBCe1-B. Additionally, by recording the intracellular pH, we found that PP1-binding consensus residues in NBCe1-B were not only critical for NBCe1-B activity, but also relevant to its surface expression level. Therefore, we reported that NBCe1-B, as a substrate of PP1, contains these residues in the C-terminal region and that the direct interaction between NBCe1-B and PP1 is functionally critical in controlling the regulation of the HCO₃⁻ transport. These results suggested that like IRBIT, PP1 was another novel regulator of HCO₃⁻ secretion in several types of epithelia.

Intracellular calcium-dependent regulation of the sperm-specific calcium-activated potassium channel, hSlo3, by the BK(Ca) activator LDD175

Plasma membrane hyperpolarization associated with activation of Ca²⁺-activated K⁺ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary γ2-subunit, LRRC52 (leucine-rich-repeat–containing 52), is known to mediate the pH-sensitive, sperm-specific K⁺ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical BK(Ca) activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting γ2 subunit, hLRRC52. As previously reported, Slo3 K⁺ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 K⁺ current, and internal alkalinization and Ca²⁺ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 K⁺ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular Ca²⁺. In contrast, elevation of intracellular Ca²⁺ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate Ca²⁺-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.

Methylene Blue-stained Interstitial Cells are Electrically Active in the Myenteric Board Freshly Prepared from the Murine Small Intestine

Many gastrointestinal muscles show electrical oscillation, so-called "slow wave", originated from interstitial cells of Cajal (ICCs). Thus, a technique to freshly isolate the cells is indispensable to explore the electrophysiological properties of the ICCs. To apply an enzyme solution on the serosal surface for cell isolation, the intestine was inverted and 0.02% trypsin solution and 0.04% collagenase solution were applied to serosal cavity. After the enzyme treatment, mucosal layer was removed and longitudinal muscle layer was gently separated from the rest of tissue. The thin layer was stretched in the recording chamber and mounted on an inverted microscope. Using beta-escine, perforated whole cell patch clamp technique was used. Under a microscope, the tissue showed smooth muscle cells and interstitial cells around the myenteric plexus. Under voltage clamp condition, three types of membrane potential were recorded. One group of interstitial cells, which were positive to methylene blue and CD34, showed spontaneous outward current. These cells had bipolar shape and were considered as fibroblast-like cells because of their peculiar shape and arrangement. Another group, positive to c-kit and methylene blue, showed spontaneous inward current. These cells had more rounded shape and processes and were considered as ICCs. The third, positive to c-kit and had granules containing methylene blue, showed quiet membrane potentials under the voltage-clamp mode. These cells appeared to be resident macrophages. Therefore, in the freshly isolated thin tissue preparation, methylene blue could easily identify three types of cells rather than morphological properties. Using this method, we were able to study electrical properties of fibroblast and residential macrophage as well as myenteric ICCs.