Progress in pharmacological research of chemokine like factor 1 (CKLF1).

Currently, the research of chemokines has penetrated into many fields of life science. A new kind of chemokines, chemokine like factor 1 (CKLF1), which is cloned through suppression subtractive hybridisation (SSH) technology is expressed widely in human body, especially in the lung and peripheral blood leukocytes. CKLF1 has a broad spectrum of chemotaxic activity for many cells, such as lymphocytes, macrophages, bone marrow cells, nerve cells and so on. In addition, CKLF1 also stimulates the regeneration of skeletal muscle cells in vivo. Collecting data derived from our and other laboratories show that CKLF1 has an important relationship with allergic diseases, autoimmune diseases, tumors, cardio-cerebrovascular diseases and so on. Therefore, there be an important theoretical purport and applied value to make a summary of pharmacological progress of CKLF1.

IMM-H004, a Novel Coumarin Derivative Compound, Inhibits H2O2-Induced Neurotoxicity via Antioxidant and Antiapoptosis in PC12 Cells.

IMM-H004 [7-hydroxy-5-methoxy-4-methyl-3-(4-methylpiperazin-1-yl)-coumarin] is a novel derivative of coumarin, which played neuroprotective roles in brain ischemia in rats in previous studies. Although antiapoptosis and improving synapsis structure were proved, the effects and mechanisms of IMM-H004 in brain ischemia need further study. In this paper, the effect of IMM-H004 on H2O2-induced neurotoxicity in pheochromocytoma (PC12) cells was researched. Morphological observation, MTT method and PI/Hoechst staining were used to indicate cell viability and apoptosis. JC-1 and DCFH-DA were used to test mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), respectively. The antioxidative activity was detected by Glutathione (GSH) and Total Antioxidant Capacity (TAC) Assay kits. Western blot was used to test apoptosis related proteins. Our results showed that treatment with 1-10 µM IMM-H004 markedly increased cell viability and decreased cell apoptosis induced by H2O2. Moreover, 1-10 µM IMM-H004 could enhance MMP and protect mitochondrial function. 1-10 µM IMM-H004 also could lower the ROS and raise the GSH and TAC level. Furthermore, 1-10 µM IMM-H004 could decrease the ratio of Bax/Bcl-2 and increase the ratio of p-AKT/AKT, which were related to apoptosis and survival. All these indicated that IMM-H004 protects PC12 cells against H2O2-induced neurotoxicity. Antioxidative and antiapoptosis may be the mechanisms of IMM-H004 in brain ischemia. These studies indicate that IMM-H004 might be a potential drug for treatment brain ischemia.

Total synthesis and neuroprotective effect of O-methylmurrayamine A and 7-methoxymurrayacine.

O-Methylmurrayamine A (7) and 7-methoxymurrayacine (8) are natural products isolated from Murraya koenigii and Murraya siamensis, respectively. In this paper, we report the synthesis of 7 and 8 which are featured in the key step of cyclization to form carbazole intermediate 5 with mild conditions. The structures were confirmed by 1H NMR, 13C NMR, and HR-ESI-MS. In addition, compounds 7 and 8 were tested for their neuroprotective effects against H2O2-induced PC12 cell damage. The results showed that compounds 7 and 8 have neuroprotective effect.

IMM-H004, A New Coumarin Derivative, Improved Focal Cerebral Ischemia via Blood-Brain Barrier Protection in Rats.

OBJECTIVE: IMM-H004 (7-hydroxy-5-methoxy-4-methyl-3-[4-methylpiperazin-1-yl]-2H-chromen-2-one) is a novel coumarin derivative that showed better effect in improving global cerebral ischemia in rats. However, the effects and mechanisms in focal cerebral ischemia were not clear. Blood-brain barrier (BBB) protection is a vital strategy for the treatment of cerebral ischemia. This study is to investigate whether IMM-H004 improves brain ischemia injury via BBB protection. METHODS: Focal brain ischemia model was induced by middle cerebral artery occlusion for 1 hour and reperfusion (MCAO/R) for 24 hours in rats. IMM-H004 (1.5, 3, 6 mg/kg) and edaravone (positive drug, 6 mg/kg) were administered after 5 minutes of occlusion. Neurological score and TTC staining were used to evaluate the effect of IMM-H004. Evans Blue (EB) staining and electron microscopy were used to assess BBB permeability. Western blot, reverse transcription-polymerase chain reaction, and immunohistochemistry were used to detect the expression of BBB structure-related proteins. RESULTS: Compared with the model group, IMM-H004 in the focal brain ischemia model improved neurological function and reduced cerebral infarction size and edema content. IMM-H004 sharply reduced the EB content and alleviated BBB structure. In addition, IMM-H004 increased the level of zonula occludens (ZO-1) and occluding, decreased the level of aquaporin 4 and matrix metalloproteinase 9, either in cortex or in hippocampus. And all of these changed were related to BBB protection. CONCLUSION: IMM-H004 improved cerebral ischemia injury via BBB protection. For a potential therapy drug of cerebral ischemia, IMM-H004 merits further study.

A new coumarin derivative, IMM-H004, attenuates okadaic acid-induced spatial memory impairment in rats.

AIM: A novel coumarin derivative 7-hydroxy-5-methoxy-4-methyl-3-(4-methylpiperazin-1-yl)-coumarin (IMM-H004) has shown anti-apoptotic, anti-inflammatory and neuroprotective activities. In this study we investigated the effects of IMM-H004 on spatial memory in rats treated with okadaic acid (OKA), which was used to imitate Alzheimer's disease (AD)-like symptoms. METHODS: SD rats were administered IMM-H004 (8 mg·kg(-1)·d(-1), ig) or donepezil (positive control, 1 mg·kg(-1)·d(-1), ig) for 25 d. On d 8 and 9, OKA (200 ng) was microinjected into the right ventricle. Morris water maze test was used to evaluate the spatial memory impairments. Tau and ß-amyloid (Aß) pathology in the hippocampus was detected using Western blot and immunohistochemistry. TUNEL staining was used to detect cell apoptosis. RESULTS: OKA-treated rats showed significant impairments of spatial memory in Morris water maze test, which were largely reversed by administration of IMM-H004 or donepezil. Furthermore, OKA-treated rats exhibited significantly increased phosphorylation of tau, deposits of Aß protein and cell apoptosis in the hippocampus, which were also reversed by administration of IMM-H004 or donepezil. CONCLUSION: Administration of IMM-H004 or donepezil protects rats against OKA-induced spatial memory impairments via attenuating tau or Aß pathology. Thus, IMM-H004 may be developed as a therapeutic agent for the treatment of AD.

Bioactive Coumarins from the Stems of Clausena emarginata.

Five new coumarins, clauemarmarins I - M (1 - 4), together with 10 known analogs (5 - 14), were isolated from the stems of Clausena emarginata. Compounds 8 - 13 were obtained from this plant for the first time. Their structures were established and elucidated by comprehensive analysis of spectroscopic data. The absolute configurations of 1 - 4 were further determined by their electronic circular dichroism spectroscopy. Compounds 5, 7, 12, and 14 exhibited inhibitory effects on LPS-induced NO production. Compounds 5 - 7 showed selective neuroprotective effects in Aß25 - 35 model at 10 µm.

[Advances in pharmacological studies of Panax notoginseng saponins on brain ischemia-reperfusion injury].

Sanqi in Chinese herbal medicine is the root and rhizoma of Panax notoginseng (Burk.) F.H. Chen, which belongs to genus Panax in the Araliaceae family and is widely used as a tonic medicine in the traditional Chinese medicine. The main active constituents of sanqi are Panax notoginseng saponins, including ginsenoside Rg1, Rb1 and notoginsenoside R1. A wide variety of pharmaceutical applications of Panax notoginseng saponins have been reported in the regulation of blood circulation system, cardiovascular system and nervous system. Ischemic stroke, the most common form of stroke, leads to a high risk of morbidity and disability, which evolves serious medical, social and economic problems. Ischemia-reperfusion injury is the most important part in the progress of ischemic stroke. Abnormal energy metabolism, disturbance of the ion metabolism, free radical injury, inflammatory reactions all participate in the complex pathological mechanisms of ischemia- reperfusion injury. Over the past few decades, substantial studies demonstrated that Panax notoginseng saponins possessed a significant protective effect on ischemia-reperfusion injury. However, little is known about the underlying mechanisms of the protective effects. In order to develop a new medicine from Panax notoginseng, we provide a review of the major literatures on the pharmaceutical actions and molecular mechanisms of Panax notoginseng and Panax notoginseng saponins in the protection of ischemia-reperfusion injury.

[Advances in pharmacological studies of Panax notoginseng saponins on brain ischemia-reperfusion injury].

Sanqi in Chinese herbal medicine is the root and rhizoma of Panax notoginseng (Burk.) F.H. Chen, which belongs to genus Panax in the Araliaceae family and is widely used as a tonic medicine in the traditional Chinese medicine. The main active constituents of sanqi are Panax notoginseng saponins, including ginsenoside Rg1, Rb1 and notoginsenoside R1. A wide variety of pharmaceutical applications of Panax notoginseng saponins have been reported in the regulation of blood circulation system, cardiovascular system and nervous system. Ischemic stroke, the most common form of stroke, leads to a high risk of morbidity and disability, which evolves serious medical, social and economic problems. Ischemia-reperfusion injury is the most important part in the progress of ischemic stroke. Abnormal energy metabolism, disturbance of the ion metabolism, free radical injury, inflammatory reactions all participate in the complex pathological mechanisms of ischemia- reperfusion injury. Over the past few decades, substantial studies demonstrated that Panax notoginseng saponins possessed a significant protective effect on ischemia-reperfusion injury. However, little is known about the underlying mechanisms of the protective effects. In order to develop a new medicine from Panax notoginseng, we provide a review of the major literatures on the pharmaceutical actions and molecular mechanisms of Panax notoginseng and Panax notoginseng saponins in the protection of ischemia-reperfusion injury.

Forsythoneosides A-D, Neuroprotective Phenethanoid and Flavone Glycoside Heterodimers from the Fruits of Forsythia suspensa.

Forsythoneosides A-D (1-4), four unusual adducts of a flavonoid unit fused to a phenylethanoid glycoside through a pyran ring or carbon-carbon bond, and four new phenylethanoid glycosides (5-8) were isolated from the fruits of Forsythia suspensa, together with nine known compounds. The structures of 1-8, including their absolute configurations, were elucidated by spectroscopic data as well as experimental and calculated electronic circular dichroism analysis. Compounds 2 and 4 inhibited PC12 cell damage induced by rotenone, and increased cell viability from 53.9 ± 7.1% to 70.1 ± 4.0% and 67.9 ± 5.2% at 0.1 µM, respectively.

4-Hydroxybenzyl-substituted glutathione derivatives from Gastrodia elata.

Seven new 4-hydroxybenzyl-substituted glutathione derivatives (2-8), together with a known analogue (1), were isolated from the aqueous extract of Gastrodia elata Blume rhizomes. Their structures were determined by using spectroscopic and chemical methods. The absolute configurations of 1-8 were assigned by using Marfey's method, combined with comparing the NMR and CD spectroscopic data of sulfoxide moieties in 3-6 with those of S-(4-hydroxybenzyl)cysteine sulfoxide stereoisomers (9-12) synthesized as authentic samples. The configurations of 9-12 were confirmed by electronic CD calculations based on the quantum-mechanical time-dependent density functional theory. Furthermore, the structures of 1, 3, 5, 7, and 8 were verified by synthesis. Compound 3 was active against serum deprivation-induced PC12 cell damage and synthetic 9-14 exhibited activity against Fe(2+)-cysteine induced rat liver microsomal lipid peroxidation.

[Neuroprotective effect screening and the mechanism of 10 kinds of coumarin derivatives].

The study reports the detection of neuroprotective effect of 10 kinds of coumarin derivatives and explores their possible mechanism. MTT method was used to screen the neuroprotective effect of 10 coumarin derivatives on neurotoxic agents (Aß25-35 and rotenone) or OGD (oxygen-glucose deprivation). A compound with better protective effect was obtained. Then the effect of this compound on neurotoxic agents on PC12 was detected by the morphological observation. Furthermore, the effect of compound 3 on microglia with lipopolysaccharide (LPS) induced inflammation was detected. And the inflammatory factor was tested. Finally, direct free radical scavenging ability was detected. Compound 3 was found to be the best compound through three neurons toxic models. Not only compound 3 ameliorated cell viability reduced by three neurons toxic models, but also significantly inhibited the production of inflammatory factor (TNF-α and IL-1ß). And its free radical scavenging ability is very good, especially the effect on superoxide anion, which is comparable with vitamin C. The significant scavenging effect of compound 3 on superoxide anion might be the mechanism of the neuroprotection. Compound 3 as a potential neural cell protective agent merits further investigation.

Development and validation of high-throughput screening assays for poly(ADP-ribose) polymerase-2 inhibitors.

Poly(ADP-ribose) polymerase-1 and -2 (PARP1/2) are two key facilitators of DNA repair and are implicated in the pathogenesis of cancers and several chronic diseases. Inhibitors of PARP1/2 have shown powerful therapeutic effects in the treatment of cancer, cerebral ischemia, and inflammation. In addition, evidence from several studies suggests unique functions for PARP2 in genome surveillance, spermatogenesis, adipogenesis, and T cell development, and PARP2-specific inhibitors might have many other applications. To acquire PARP1/2 inhibitors, many high-throughput screening (HTS) assays for PARP1 inhibitors have been developed. However, detailed screening assays for PARP2 inhibitors have not been reported. Herein, three HTS assays for PARP2 inhibitors were developed and validated with reference inhibitors in each case. The results suggest that the HTS assays for PARP2 inhibitors using chemical quantification of NAD(+), biotin-based quantification of PAR, and ELISA quantification of PAR are sensitive, robust, and cost effective.

Iridal-type triterpenoids with neuroprotective activities from Iris tectorum.

Six novel iridal-type triterpenoids with a previously unreported 3,6-dihydro-2H-pyran moiety, named spirioiridotectals A-F (1-6), were isolated from the ethanol extract of the rhizomes of Iris tectorum. Their structures were elucidated on the basis of extensive spectroscopic analysis. Furthermore, in in vitro bioactivity assays, compounds 1, 2, and 6 exhibited neuroprotective activities against serum-deprivation-induced PC12 cell damage.

Nrf2 pathway activation contributes to anti-fibrosis effects of ginsenoside Rg1 in a rat model of alcohol- and CCl4-induced hepatic fibrosis.

AIM: To investigate the anti-fibrosis effects of ginsenoside Rg1 on alcohol- and CCl4-induced hepatic fibrosis in rats and to explore the mechanisms of the effects. METHODS: Rats were given 6% alcohol in water and injected with CCl4 (2 mL/kg, sc) twice a week for 8 weeks. Rg1 (10, 20 and 40 mg/kg per day, po) was administered in the last 2 weeks. Hepatic fibrosis was determined by measuring serum biochemical parameters, HE staining, Masson's trichromic staining, and hydroxyproline and α-SMA immunohistochemical staining of liver tissues. The activities of antioxidant enzymes, lipid peroxidation, and Nrf2 signaling pathway-related proteins (Nrf2, Ho-1 and Nqo1) in liver tissues were analyzed. Cultured hepatic stellate cells (HSCs) of rats were prepared for in vitro studies. RESULTS: In the alcohol- and CCl4-treated rats, Rg1 administration dose-dependently suppressed the marked increases of serum ALT, AST, LDH and ALP levels, inhibited liver inflammation and HSC activation and reduced liver fibrosis scores. Rg1 significantly increased the activities of antioxidant enzymes (SOD, GSH-Px and CAT) and reduced MDA levels in liver tissues. Furthermore, Rg1 significantly increased the expression and nuclear translocation of Nrf2 that regulated the expression of many antioxidant enzymes. Treatment of the cultured HSCs with Rg1 (1 µmol/L) induced Nrf2 translocation, and suppressed CCl4-induced cell proliferation, reversed CCl4- induced changes in MDA, GPX, PCIII and HA contents in the supernatant fluid and α-SMA expression in the cells. Knockdown of Nrf2 gene diminished these actions of Rg1 in CCl4-treated HSCs in vitro. CONCLUSION: Rg1 exerts protective effects in a rat model of alcohol- and CCl4-induced hepatic fibrosis via promoting the nuclear translocation of Nrf2 and expression of antioxidant enzymes.

Lung ultrasound to diagnose infectious pneumonia of newborns: A prospective multicenter study.

INTRODUCTION: Whether lung ultrasound (LUS) can be used for pathogenic diagnosis remains controversial. This study was conducted to clarify whether ultrasound has diagnostic value for etiology. METHODS: A total of 135 neonatal pneumonia patients with an identified pathogen were enrolled from the newborn intensive care units of 10 tertiary hospitals in China. The study ran from November 2020 to December 2021. The infants were divided into various groups according to pathogens, time of infection, gestational age, and disease severity. The distribution of pleural line abnormalities, B-line signs, and pulmonary consolidation, as well as the incidence of air bronchogram and pleural effusion based on LUS, were compared between these groups. RESULTS: There were significant differences in pulmonary consolidation. The sensitivity and specificity of the diagnosis of severe pneumonia based on the extent of pulmonary consolidation were 83.3% and 85.2%, respectively. The area under the receiver operating characteristic curve for the identification of mild or severe pneumonia based on the distribution of pulmonary consolidation was 0.776. CONCLUSION: LUS has good performance in diagnosing and differentiating the severity of neonatal pneumonia but cannot be used for pathogenic identification in the early stages of pneumonia.

IMM-H004 reduced okadaic acid-induced neurotoxicity by inhibiting Tau pathology in vitro and in vivo.

This study aimed to explore effects and mechanisms of 004 (IMM-H004), a novel coumarin derivative, in OKA (okadaic acid)-induced AD (Alzheimer's disease)-like model. In vitro, MTT, LDH, and Annexin V/FITC flow cytometry assay were used to test cell survival. In vivo, OKA microinjection was conducted to simulate AD-like neuropathology. Morris water maze and Nissl staining were used to detect spatial memory function and neuronal damage respectively. Western blot and immunohistochemistry were used to study the mechanisms of 004 in Tau pathology. The results showed that 004 reduced cell death and increased survival in PC12 cells, and decreased neuronal injury in the hippocampus in rats. 004 improved learning and memory functions in OKA-treated rats. The mechanistic studies indicated that 004 inhibited phosphorylation of Tau protein by down-regulating the activity of protein kinases CDK5 and GSK3ß and increasing PP2A activity. Overall, 004 improved spatial memory impairments and neuron cells injury induced by OKA; on the other hand, 004 inhibited Tau hyperphosphorylation by regulating CDK5, GSK3ß and PP2A.

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat's weight (260-330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa's score, a larger infarct volume and a greater model success ratio were screened using the Longa's score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

Metabolism of IMM-H004 and Its Pharmacokinetic-Pharmacodynamic Analysis in Cerebral Ischemia/Reperfusion Injured Rats.

IMM-H004, a derivative of coumarin, is a promising candidate for the treatment of cerebral ischemia. The pharmacodynamic mechanisms of IMM-H004 are still under exploration. The present study was conducted to explore the pharmacoactive substances of IMM-H004 from the perspective of drug metabolism. Four metabolites of IMM-H004 including demethylated metabolites M1 and M2, glucuronide conjugate IMM-H004G (M3), and sulfated conjugate M4 were found in rats in vivo. IMM-H004G was the major metabolite in rats and cultured human hepatocytes, and uridine diphosphate-glucuronosyltransferase (UGT) was found to catalyze the metabolism of IMM-H004 in human liver microsomes (HLMs) and rat liver microsomes (RLMs) with high capacity (V max at 3.25 and 5.04 nmol/min/mg protein). Among 13 recombinant human UGT isoforms, UGT1A7, 1A9, 1A8, and 1A1 appeared to be primarily responsible for IMM-H004G formation. The exposure and duration of IMM-H004G (28,948 h × ng/ml of area under the plasma concentration-time curve (AUC), 6.61 h of t 1/2ß) was much higher than that of the parent drug (1,638 h × ng/ml of AUC, 0.42 h of t 1/2ß) in transient middle cerebral artery occlusion/reperfusion (MCAO/R) rats, consistent with the malondialdehyde (MDA) inhibition effect for at least 10 h. Further pharmacological study revealed that IMM-H004G exhibited a similar neuroprotective activity to that of the parent drug on both oxygen-glucose deprivation injured PC12 cells and transient MCAO/R injured rats. These results demonstrate that both prototype and IMM-H004G are the active pharmaceutical substances, and IMM-H004G, at least in part, contributes to the maintenance of anti-cerebral ischemia efficacy of IMM-H004.

Validation of a preclinical animal model to assess brain recovery after acute stroke.

This study was to validate the animal model for the research in the stage of recovery and sequela of ischemic stroke. For its recognized many advantages and widespread applications, middle cerebral artery occlusion / reperfusion (MCAO/R) in Male Sprague-Dawley rats was chosen to be the foundation model. Then the weight of rats (260-330 g), the thread bolt type (2636/2838/3040/3043), the time of brain infarct (2/3 h) were tested to choose the larger infarct volume, higher Longa's score and model success rate through Longa's score and TTC staining. Finally, optimum condition of model was used in long period observing from 1 to 90 days after MCAO/R via five assessment of sensorimotor functions and TTC staining. The results showed that the optimal rat model of cerebral infarction in the stage of recovery and sequela of ischemic stroke maybe the model rats which were 300 g weight and MCAO with 3040 line-lock for 3 h before reperfusion. In these conditions, the Longa's score was 2.1 ±â€¯0.2, and infarct volume was 23.0 ±â€¯2.4%. The sensorimotor functional test of bilateral asymmetry had significant difference from 1 to 90 days, the test of grid-walking had significant difference from 1 to 60 days, while other tests had significant difference only at 1 day after MCAO/R. In conclusion, 3040-300 g-3 h was the most appropriate condition, and the appropriate index of sensorimotor functions were bilateral asymmetry and grid-walking test.

Pyrano[3,2-a]carbazole alkaloids as effective agents against ischemic stroke in vitro and in vivo.

A series of pyrano[3,2-a]carbazole alkaloids were designed and synthesized as analogues of Claulansine F (Clau F, 10a) isolated from Clausena lansium. Some of compounds showed strong neuroprotective effects and were promising agents against ischemic stroke. Among these compounds, 7c was the most active in inhibiting the programmed death of PC12 cells and primary cortical neurons. This compound induced neuroprotection following ischemic reperfusion and decreased neurological deficit scores in treated animals. Furthermore, 7c could penetrate the blood-brain barrier (BBB) in rats, and its exposure in the brain was 4.3-fold higher than that in plasma. More importantly, compared to edaravone, 7c exhibited stronger free radical scavenging activity. Our findings suggest that 7c may be promising for further evaluation as an intervention for ischemic stroke.