[Determination of 13 chemical components of Epimedii Folium in pharmacopoeia by UPLC method combined with quantitative analysis of multicomponents by single-marker].

The quantitative analysis of multicomponents by single-marker(QAMS) was established for 13 chemical components of Epimedii Folium, including neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuoside Ⅰ, so as to investigate the feasibility and accuracy of this method in evaluating the quality of Epimedii Folium materials from different origins and different varieties. Through the scientific and accurate investigation of the experimental method, the external standard method was used to determine the content of 13 chemical components in epimedium brevieornu. At the same time, icariin was used as the internal standard, and the relative correction factors of icariin with neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuoside Ⅰ were established, respectively. The contens of neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuosideⅠ in Epimedii Folium were calculated by QAMS. Finally, the difference between the measured value and the calculated value was compared to verify the accuracy and scientific nature of QAMS in the determination. The relative correction factor of each component had better repeatability, and there was no significant difference between the results of the external standard method and those of QAMS. With icariin as the internal standard, QAMS simultaneously determining neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuoside Ⅰ can be used for quantitative analysis of Epimedii Folium.

Anti-apoptotic effect of adrenomedullin gene delivery on Leydig cells by suppressing TGF-ß1 via the Hippo signaling pathway.

This study aims to establish whether adrenomedullin (ADM) is capable to restore the steroidogenic functions of Leydig cells by suppressing transforming growth factor-ß1 (TGF-ß1) through Hippo signaling. Primary Leydig cells were treated with lipopolysaccharide (LPS), an adeno-associated virus vector that expressed ADM (Ad-ADM) or sh-RNA of TGF-ß1 (Ad-sh-TGF-ß1). The cell viability and medium concentrations of testosterone were detected. Gene expression and protein levels were determined for steroidogenic enzymes, TGF-ß1, RhoA, YAP, TAZ and TEAD1. The role of Ad-ADM in the regulation of TGF-ß1 promoter was confirmed by ChIP and Co-IP. Similar to Ad-sh-TGF-ß1, Ad-ADM mitigated the decline in the number of Leydig cells and plasma concentrations of testosterone by restoring the gene and protein levels of SF-1, LRH1, NUR77, StAR, P450scc, 3ß-HSD, CYP17 and 17ß-HSD. Similar to Ad-sh-TGF-ß1, Ad-ADM not only inhibited the LPS-induced cytotoxicity and cell apoptosis but also restored the gene and protein levels of SF-1, LRH1, NUR77, StAR, P450scc, 3ß-HSD, CYP17 and 17ß-HSD, along with the medium concentrations of testosterone in LPS-induced Leydig cells. Like Ad-sh-TGF-ß1, Ad-ADM improved LPS-induced TGF-ß1 expression. In addition, Ad-ADM suppressed RhoA activation, enhanced the phosphorylation of YAP and TAZ, reduced the expression of TEAD1 which interacted with HDAC5 and then bound to TGF-ß1 gene promoter in LPS-exposed Leydig cells. It is thus suspected that ADM can exert anti-apoptotic effect to restore the steroidogenic functions of Leydig cells by suppressing TGF-ß1 through Hippo signaling.

Local adrenomedullin gene delivery inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo.

Adrenomedullin (ADM) has beneficial effects on Leydig cells under pathological conditions, including lipopolysaccharide (LPS)-induced orchitis. Our previous studies demonstrated that ADM exerts a restorative effect on steroidogenesis in LPS-treated primary rat Leydig cells by attenuating oxidative stress, inflammation and apoptosis. In this study, we aim to investigate whether ADM inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo. Rats were administered with LPS and injected with Ad-ADM, an adeno-associated virus vector that expressed ADM. Then, rat testes were collected for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) immunofluorescence staining. Steroidogenic enzymes or steroidogenic regulatory factors or protein, including steroidogenic factor-1 (SF-1), liver receptor homologue-1 (LRH1), Nur77, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc), 3ß-HSD, cytochrome P450 17α-hydroxylase/17, 20 lyase (CYP17) and 17ß-hydroxysteroid dehydrogenase (17ß-HSD), were detected via gene expression profiling and western blot analysis. Plasma testosterone concentrations were measured. Results showed that ADM may inhibit Leydig cell dysfunction by rescuing steroidogenic enzymes and steroidogenic regulatory factors in vivo. The reduction in the number of Leydig cells after LPS exposure was reversed by ADM. ADM rescued the gene or protein levels of SF-1, LRH1, Nur77, StAR, P450scc, 3ß-HSD, CYP17 and 17ß-HSD and plasma testosterone concentrations. To summarize ADM could rescue some important steroidogenic enzymes, steroidogenic regulatory factors and testosterone production in Leydig cells in vivo.

Microglial ablation does not affect opioid-induced hyperalgesia in rodents.

ABSTRACT: Opioids are the frontline analgesics in pain management. However, chronic use of opioid analgesics causes paradoxical pain that contributes to the decrease of their efficacy in pain control and the escalation of dose in long-term management of pain. The underling pathogenic mechanism is not well understood. Microglia have been commonly believed to play a critical role in the expression of opioid-induced hyperalgesia in animal models. We performed microglial ablation experiments using either genetic (CD11b-diphtheria toxin receptor transgenic mouse) or pharmacological (colony-stimulating factor-1 receptor inhibitor PLX5622) approaches. Surprisingly, ablating microglia using these specific and effective approaches did not cause detectable impairment in the expression of hyperalgesia induced by morphine. We confirmed this conclusion with a behavioral test of mechanical and thermal hyperalgesia, in male and female mice, and with different species (mouse and rat). These findings raise caution about the widely assumed contribution of microglia to the development of opioid-induced hyperalgesia.

Case selection and implementation of tubeless percutaneous nephrolithotomy.

BACKGROUND: The tubeless percutaneous nephrolithotomy (PCNL) was proposed to eliminate the side effects of the nephrostomy tube in recent years, such as pain, channel infection, postoperative bleeding, and longer hospital stay. But there is neither clinical guidelines nor consensus about tubeless PCNL in clinical practice. The study is aimed to how to implement the tubeless PCNL step by step, including case selection preoperatively, improving the technique of the surgeon, making the correct decisions at the end of the procedure, which had not been previously examined. METHODS: From January 2017 to March 2018, 364 consecutive patients requiring PCNL were comprehensively analyzed preoperatively and patients were selected for scheduled tubeless PCNL based on four aspects. The selected patients were divided into two groups according to whether the nephrostomy tube was finally placed. The mean operative time, intraoperative blood loss, stone clearance rate, visual pain score, postoperative hospitalization days and perioperative complications were all evaluated. RESULTS: Based on the preoperative evaluation, 42 patients were selected for tubeless PCNL, among which there were finally 37 cases of completed tubeless PCNL. Compared with patients undergoing conventional PCNL, there were not statistical differences in the mean operative time (P=0.207) or intraoperative blood loss (P=0.450) in the tubeless group. Stone clearance rate was 100% in both groups. The visual pain scores in the tubeless PCNL group were lower on operation day (P=0.029), first postoperative day (P<0.001) and the day of discharge (P=0.025). The postoperative hospitalization for the tubeless PCNL group was shorter than that of the control group (P<0.001). No significant difference in grade 1 complications was seen (P=0.424), and no grade 2 or higher complications were observed in either group. CONCLUSIONS: Postoperative pain was significantly relieved and postoperative hospitalization was significantly shortened in the tubeless PCNL group. Tubeless PCNL is safe if patients are carefully selected using four criteria before operation, attention is paid to four key points and five confirmations are made during operation.

Deep Genomic Sequencing of Bladder Urothelial Carcinoma in Southern Chinese Patients: A Single-Center Study.

OBJECTIVE: Bladder urothelial carcinoma (BUC) is a common urological malignancy with molecular heterogeneity. However, the genetic feature of Chinese BUC patients is still not well-identified. METHODS: We performed deep sequencing by a large panel (450 genes) on 22 BUC samples and using matched normal bladder tissue as control. Genomic alterations (GAs), pathways and Tumor Mutation Burden (TMB) were investigated. RESULTS: The frequencies of GAs (TERT, 54.5%; CREBBP, 27.3%; GATA3, 22.7%; BRAF, 18.2%; TEK, 18.2% and GLI1, 18.2%) were significantly higher in Chinese than Western BUC patients. Other GAs' frequencies were in accordance with previous study (TP53, 50.0%; KDM6A, 31.8%; KMT2D, 22.7%; etc.). Besides, we detected gene amplification in ERBB2, FRS2, FAS, etc. The gene fusion/rearrangement took place in the chromosome 11, 12, 14, 17, 19, 22, and Y. Other than cell cycle and PI3K-AKT-mTOR, mutated genes were more associated with the transcription factor, chromatin modification signaling pathways. Interestingly, the TMB value was significantly higher in the BUC patients at stages T1-T2 than T3-T4 (P = 0.025). CONCLUSION: Deep genomic sequencing of BUC can provide new clues on the unique GAs of Chinese patients and assist in therapeutic decision.

Nonadiabatic dynamics simulation of photoinduced ring-opening reaction of 2(5H)-thiophenone with internal conversion and intersystem crossing.

In the present work, the quantum trajectory mean-field approach, which is able to overcome the overcoherence problem, was generalized to simulate internal conversion and intersystem crossing processes simultaneously. The photoinduced ring-opening and subsequent rearrangement reactions of isolated 2(5H)-thiophenone were studied based on geometry optimizations on critical structures and nonadiabatic dynamics simulations using this method. Upon 267 nm irradiation, the molecule is initially populated in the 1ππ* state. After a sudden rupture of one C-S bond within 100 fs in this state, the lowest two singlet excited states and the lowest two triplet excited states become quasi-degenerated, and then the intersystem crossing processes between singlet and triplet states accompanied by rearrangement reactions can be observed several times. Compared with our previous nonadiabatic simulations in the absence of intersystem crossing (ChemPhotoChem, 2019, 3, 897-906), some new nonadiabatic relaxation pathways involving triplet states and different ring-opening products were identified. The present work provides new mechanistic insights into the photoinduced ring-opening of thio-substituted heterocyclic molecules and reveals the importance of nonadiabatic dynamics simulation that is able to deal with multiple electronic states with different spin multiplicities.

Normalization of magnesium deficiency attenuated mechanical allodynia, depressive-like behaviors, and memory deficits associated with cyclophosphamide-induced cystitis by inhibiting TNF-α/NF-κB signaling in female rats.

BACKGROUND: Bladder-related pain symptoms in patients with bladder pain syndrome/interstitial cystitis (BPS/IC) are often accompanied by depression and memory deficits. Magnesium deficiency contributes to neuroinflammation and is associated with pain, depression, and memory deficits. Neuroinflammation is involved in the mechanical allodynia of cyclophosphamide (CYP)-induced cystitis. Magnesium-L-Threonate (L-TAMS) supplementation can attenuate neuroinflammation. This study aimed to determine whether and how L-TAMS influences mechanical allodynia and accompanying depressive symptoms and memory deficits in CYP-induced cystitis. METHODS: Injection of CYP (50 mg/kg, intraperitoneally, every 3 days for 3 doses) was used to establish a rat model of BPS/IC. L-TAMS was administered in drinking water (604 mg·kg-1·day-1). Mechanical allodynia in the lower abdomen was assessed with von Frey filaments using the up-down method. Forced swim test (FST) and sucrose preference test (SPT) were used to measure depressive-like behaviors. Novel object recognition test (NORT) was used to detect short-term memory function. Concentrations of Mg2+ in serum and cerebrospinal fluid (CSF) were measured by calmagite chronometry. Western blot and immunofluorescence staining measured the expression of tumor necrosis factor-α/nuclear factor-κB (TNF-α/NF-κB), interleukin-1ß (IL-1ß), and N-methyl-D-aspartate receptor type 2B subunit (NR2B) of the N-methyl-D-aspartate receptor in the L6-S1 spinal dorsal horn (SDH) and hippocampus. RESULTS: Free Mg2+ was reduced in the serum and CSF of the CYP-induced cystitis rats on days 8, 12, and 20 after the first CYP injection. Magnesium deficiency in the serum and CSF correlated with the mechanical withdrawal threshold, depressive-like behaviors, and short-term memory deficits (STMD). Oral application of L-TAMS prevented magnesium deficiency and attenuated mechanical allodynia (n = 14) and normalized depressive-like behaviors (n = 10) and STMD (n = 10). The upregulation of TNF-α/NF-κB signaling and IL-1ß in the L6-S1 SDH or hippocampus was reversed by L-TAMS. The change in NR2B expression in the SDH and hippocampus in the cystitis model was normalized by L-TAMS. CONCLUSIONS: Normalization of magnesium deficiency by L-TAMS attenuated mechanical allodynia, depressive-like behaviors, and STMD in the CYP-induced cystitis model via inhibition of TNF-α/NF-κВ signaling and normalization of NR2B expression. Our study provides evidence that L-TAMS may have therapeutic value for treating pain and comorbid depression or memory deficits in BPS/IC patients.

Adrenomedullin alleviates the pyroptosis of Leydig cells by promoting autophagy via the ROS-AMPK-mTOR axis.

Adrenomedullin (ADM) exerts anti-oxidant, anti-inflammatory and anti-apoptotic effects in Leydig cells. However, the role and mechanism of ADM in the pyroptosis of Leydig cells are poorly understood. This study first showed the protective effects of ADM on the pyroptosis and biological functions of Leydig cells exposed to lipopolysaccharide (LPS) by promoting autophagy. Primary rat Leydig cells were treated with various concentrations of LPS and ADM, together with or without N-acetyl-L-cysteine (NAC) or 3-methyladenine (3-MA). Cell proliferation was detected through CCK-8 and BrdU incorporation assays, and ROS level was measured with the DCFDA assay. Real-time PCR, western blot, immunofluorescence, transmission electron microscopy, TUNEL and flow cytometry were performed to examine ADM's effect on the pyroptosis, autophagy and steroidogenic enzymes of Leydig cells and AMPK/mTOR signalling. Like NAC, ADM dose-dependently reduced LPS-induced cytotoxicity and ROS overproduction. ADM also dose-dependently ameliorated LPS-induced pyroptosis by reversing the increased expression of NLRP3, ASC, caspase-1, IL-1ß, IL-18, GSDMD, caspase-3, caspase-7, TUNEL-positive and PI and active caspase-1 double-stained positive rate, DNA fragmentation and LDH concentration, which could be rescued via co-incubation with 3-MA. ADM dose-dependently increased autophagy in LPS-induced Leydig cells, as confirmed by the increased expression of LC3-I/II, Beclin-1 and ATG-5; decreased expression of p62 and autophagosomes formation; and increased LC3-II/LC3-I ratio. However, co-treatment with 3-MA evidently decreased autophagy. Furthermore, ADM dose-dependently rescued the expression of steroidogenic enzymes, including StAR, P450scc, 3ß-HSD and CYP17, and testosterone production in LPS-induced Leydig cells. Like rapamycin, ADM dose-dependently enhanced AMPK phosphorylation but reduced mTOR phosphorylation in LPS-induced Leydig cells, which could be rescued via co-incubation with 3-MA. In addition, pyroptosis was further decreased, and autophagy was further promoted in LPS-induced Leydig cells upon co-treatment with ADM and rapamycin. ADM may protect the steroidogenic functions of Leydig cells against pyroptosis by activating autophagy via the ROS-AMPK-mTOR axis.

Neuregulin-1-ErbB signaling promotes microglia activation contributing to mechanical allodynia of cyclophosphamide-induced cystitis.

AIMS: Central sensitization playsimportant roles in cyclophosphamide (CYP)-induced cystitis. In addition, as a visceral pain, CYP-induced chronic pain shares common pathophysiological mechanisms with neuropathic pain. Previous studies demonstrated that neuregulin-1 (Nrg1)-ErbB signaling contributes to neuropathic pain, but whether and how this signaling influences mechanical allodynia in CYP-induced cystitis is unclear. This study aimed to determine whether and how Nrg1-ErbB signaling modulates mechanical allodynia in a CYP-induced cystitis rat model. METHODS: Systemic injection with CYP was used to establish a rat model of bladder pain syndrome/interstitial cystitis (BPS/IC). An irreversible ErbB family receptor inhibitor, PD168393, and exogenous Nrg1 were intrathecally injected to modulate Nrg1-ErbB signaling. Mechanical allodynia in the lower abdomen was assessed with von-Frey filaments using the up-down method. Western blot analysis and immunofluorescence staining were used to measure the expression of Nrg1-ErbB signaling, Iba-1, p-p38, and IL-1ß in the L6-S1 spinal dorsal horn (SDH). RESULTS: We observed upregulation of Nrg1-ErbB signaling as well as overexpression of the microglia activation markers Iba-1 and p-p38 and the proinflammatory factor, interleukin-1ß (IL-1ß), in the SDH of the cystitis group. Further, treatment with PD168393 attenuated mechanical allodynia in CYP-induced cystitis and inhibited microglia activation, leading to decreased production of IL-1ß. The inhibitor PD168393 reversed the algesic effect of exogenous Nrg1 on the cystitis model. CONCLUSIONS: Nrg1-ErbB signaling may promote microglia activation, contributing to mechanical allodynia of CYP-induced cystitis. Our study showed that modulation of Nrg1-ErbB signaling may have therapeutic value for treating pain symptoms in BPS/IC.

Increased severity of inflammation correlates with elevated expression of TRPV1 nerve fibers and nerve growth factor on interstitial cystitis/bladder pain syndrome.

OBJECTIVES: Although evidence supports a role for inflammation in interstitial cystitis/bladder pain syndrome (IC/BPS), the mechanism remains unknown. We determined whether inflammation causes an elevated expression of nerve growth factor (NGF) and transient receptor potential vanilloid receptor subtype 1 (TRPV1) and correlated them with the symptoms. METHODS: Bladder biopsies were obtained from 53 IC/BPS patients and 27 controls, and hematoxylin and eosin staining, immunostaining and Western blotting were performed to detect inflammation, TRPV1-immunoreactive and PGP9.5-immunoreactive nerve fibers, and NGF, respectively. Symptoms were assessed using the Pelvic Pain/Urgency/Frequency (PUF) questionnaire and pain visual analogue scale scores. Suburothelial nerve fiber density was quantified and correlated with PUF scores. RESULTS: Increased severity of inflammation was correlated with a higher TRPV1-immunoreactive nerve fiber density (r = 0.4113, p = 0.0024) and higher NGF levels (r = 0.3775, p = 0.0052). Suburothelial TRPV1-immunoreactive nerve fiber density was significantly correlated with pain scores and urgency scores (r = 0.3320, p = 0.0145 and r = 0.3823, p = 0.0039, respectively). PGP9.5-immunoreactive nerve fibers were significantly increased in IC/BPS (p = 0.0193) and had a positive relationship with inflammation severity (r = 0.6138, p < 0.0001). CONCLUSIONS: Our study revealed increased severity of inflammation correlated with a higher expression of TRPV1-immunoreactive nerve fibers and NGF in IC/BPS and correlated with clinical symptoms.