YZG-330 regulates the TRPM8 ion channel through the P38 MAPK signaling pathway to reduce body temperature in mice / 药学学报

Preliminary research in our laboratory found that compound YZG-330 can reduce mouse body temperature, which could be blocked by adenosine A1 receptor (A1R) antagonist DPCPX. Based on the downstream signaling pathway of the A1R, the mechanism by which YZG-330 lowers body temperature was further studied. The pharmacodynamics of YZG-330 was evaluated by measuring the rectal temperature; expression of the transient receptor potential (TRP) ion channel, the P38 protein and its phosphorylated form in mouse hypothalamic homogenate were detected by Western blotting. A Ca2+ fluorescent probe, Fluo-3AM, was added to cells to detect the effect of YZG-330 on the Ca2+ content of mouse hypothalamic cells. YZG-330 dose-dependently reduced the body temperature in mice, and the selective P38 inhibitor SB-203580 (20 mg·kg-1, i.p.) significantly inhibited the hypothermic effect of YZG-330. A TRPM8 antagonist 2 (0.1 μg per mouse, i.c.v.) markedly attenuated the hypothermic effect of YZG-330 (0.25 or 1 mg·kg-1, i.p.). YZG-330 (2 mg·kg-1, i.p.) significantly increased the phosphorylation of P38, an effect that could be attenuated by the A1R antagonist DPCPX (5 mg·kg-1, i.g.) in mouse hypothalamus. In addition, YZG-330 also prominently enhanced the expression of TRPM8, which could be blocked by SB-203580; YZG-330 (0.1-10 μmol·L-1) increased intracellular Ca2+ concetration in mouse hypothalamic cells in a dose-dependent manner, and was inhibited by the A1R inhibitor DPCPX (0.5 and 1 μmol·L-1) and TRPM8 antagonist 2 (1 μmol·L-1). In conclusion, YZG-330 exerts its hypothermic effect by activating the A1R to promote the phosphorylation of P38 protein and thereby up-regulating the expression and activity of the TRPM8 ion channel, resulting in increased intracellular Ca2+ concentration to stimulate mouse hypothalamus cells to down-regulate body temperature. All animal experiments were approved by the Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences.

Spinal transcription factor GATA4 mediated adenosine A1 receptor activation contributes to electroacupuncture analgesia in neuropathic pain rats / 针刺研究

OBJECTIVE: To investigate the effect of electroacupuncture (EA) on pain behaviors and expression of spinal transcription factor GATA-binding Protein 4 (GATA4) and adenosine A1 receptor in neuropathic pain rats, so as to explore its mechanism underlying pain relief. METHODS: The present study includes 2 parts. In the first part, 18 SD rats were randomly divided into control, adenovirus short-hairpin interference RNA for GATA4 (AV-shGATA4 RNA) and adenovirus empty vector (AV-control short-hairpin RNA, AV-shCTRL) groups, with 6 rats in each group. The expression of GATA4 protein in the lumbar spinal cord (L4-L6) was detected to evaluate the transfection efficiency of AV-shGATA4 RNA (silencing GATA4 expression). In the second part, thirty SD rats were randomly divided into 5 groups, namely sham operation, CCI model, EA, EA+AV-shGATA4 RNA, and EA+AV-shCTRL groups, with 6 rats in each group. The neuropathic pain model was established by chronic constriction injury (CCI) of the right sciatic nerve. On the 7th day following modeling, EA was applied to the right "Zusanli"(ST36) and "Taichong"(LR3) (1 mA，2 Hz /100 Hz) for 30 min. Rats of the EA+AV-shGATA4 RNA and EA+AV-shCTRL groups received intrathecal injection of AV-shGATA4 RNA and AV-shCTRL(1×1011 PFU/mL，10 μL)at the spinal L4-L6 segments, separately, 48 h before EA intervention. The mechanical pain threshold and thermal pain threshold of the affected limb were detected before molding, 7 days following molding and 60 min after EA. The expressions of adenosine A1 receptor and GATA4 protein in the spinal cord (L4-L6) were detected by Western blot. RESULTS: Outcomes of the first part showed that compared with the control group, no significant changes were found in the mechanical and thermal pain thresholds in both AV-shCTRL and AV-shGATA4 RNA groups and in the expression of spinal GATA4 protein of the AV-shCTRL group (P＞0.05). The expression of spinal GATA4 protein of the AV-shGATA4 RNA group was significantly lower than that of the AV-shCTRL group (P 0.05). On the 7th day following modeling, the mechanical and thermal pain thresholds were significantly lowered in compa-rison with their own pre-modeling of each group and with the sham operation group (P0.05), suggesting a critical involvement of GATA4 in EA analgesia. The expression levels of adenosine A1 receptor and GATA4 protein were significantly increased in the model group than in the sham operation group (P0.05), suggesting that the effects of EA in up-regulating the expression of A1 receptor and GATA4 were eliminated after silencing GATA4 protein. CONCLUSION: EA of ST36 and LR3 can relieve pain by increasing the expression of adenosine A1 receptor of the lumbar spinal cord in neuropathic pain rats, which is probably mediated by GATA4 protein.

The research progress on the mechanism of adenosine A1 receptor-mediated calcitonin gene-related peptide to relieve migraine

Currently, the pathogenesis of migraine is unclear. The trigeminal vascular reflex theory is the dominant pathogenesis theory, and its core parts are neurogenic inflammation and pain sensitisation. Calcitonin gene related peptide (CGRP) is the most powerful vasodilating peptide in brain circulation. It is also a marker of trigeminal nerve microvascular activation that plays a synergistic role in the pathogenesis of migraine. Adenosine A1 receptor (A1R) can inhibit the release of CGRP in the trigeminal nerve vascular system to alleviate migraine by mediating adenosine. This review summarises the progress of research on the alleviation of migraine by using A1R-mediated CGRP.

Adenosine a1 receptor agonist protects against hippocampal neuronal injury after lithium chloride-pilocarpine-induced epilepsy

Abstract Background Adenosine A1 receptor (AA1R) is widely present in the central nervous system, exerting brain protective antiepileptic effects, mainly by binding corresponding G proteins. We evaluated the neuroprotective effects of AA1R on hippocampal neuronal injury after lithium chloride-pilocarpine-induced epilepsy in rats. Materials and Methods A total of 60 male SD rats were randomly divided into four groups (n = 15/group): normal control, epilepsy, epilepsy + AA1R antagonist (DPCPX), and epilepsy + AA1R agonist (2-CAdo). An epilepsy model was established through kindling by lithium chloride-pilocarpine. The four groups were observed on days 1, 14, and 30. Pathological and morphological changes of hippocampal neurons were observed by HE staining; apoptosis was detected by TUNEL assay. Caspase-3 and GABA receptor expressions were detected by Western blot. Results In the hippocampal CA3 area of the epilepsy group, the cellular structure was not neatly arranged, and some neurons were swelling, thick, and incomplete. Compared with the epilepsy group at the same time point, cells in the epilepsy + DPCPX group had an increased distortion, disorganization, edema, cytoplasmic vacuoles, and degeneration. In the epilepsy + 2-CAdo group, cell arrangement was regular and orderly, and structural damages were lessened. Compared with the normal control group at the same time point, the epilepsy group underwent evident neuronal apoptosis, with a significantly higher apoptotic index (AI) (p < 0.05). Compared with the epilepsy group, the neuronal apoptosis of the epilepsy + DPCPX group was boosted, and the AI significantly increased (p < 0.05). The neuronal apoptosis of the epilepsy + 2-CAdo group was inhibited, and the AI significantly decreased (p < 0.05). Compared with the epilepsy group, the caspase-3 expression levels of the epilepsy + DPCPX group on days 14 and 30 were significantly upregulated (p < 0.05), but those of the epilepsy + 2-CAdo group were significantly downregulated (p < 0.05). Conclusions AA1R abated cell edema and reduced apoptosis, exerting neuroprotective effects on hippocampal neuronal injury after lithium chloride-pilocarpine-induced epilepsy.

Sleep Promoting Effect of Luteolin in Mice via Adenosine A1 and A2A Receptors

Luteolin, a widespread flavonoid, has been known to have neuroprotective activity against various neurologic diseases such as epilepsy, and Alzheimer’s disease. However, little information is available regarding the hypnotic effect of luteolin. In this study, we evaluated the hypnotic effect of luteolin and its underlying mechanism. In pentobarbital-induced sleeping mice model, luteolin (1, and 3 mg/kg, p.o.) decreased sleep latency and increased the total sleep time. Through electroencephalogram (EEG) and electromyogram (EMG) recording, we demonstrated that luteolin increased non-rapid eye movement (NREM) sleep time and decreased wake time. To evaluate the underlying mechanism, we examined the effects of various pharmacological antagonists on the hypnotic effect of luteolin. The hypnotic effect of 3 mg/kg of luteolin was not affected by flumazenil, a GABAA receptor-benzodiazepine (GABAAR-BDZ) binding site antagonist, and bicuculine, a GABAAR-GABA binding site antagonist. On the other hand, the hypnotic effect of 3 mg/kg of luteolin was almost completely blocked by caffeine, an antagonist for both adenosine A1 and A2A receptor (A1R and A2AR), 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), an A1R antagonist, and SCH-58261, an A2AR antagonist. From the binding affinity assay, we have found that luteolin significantly binds to not only A1R but also A2AR with IC₅₀ of 1.19, 0.84 μg/kg, respectively. However, luteolin did not bind to either BDZ-receptor or GABAAR. From these results, it has been suggested that luteolin has hypnotic efficacy through A1R and A2AR binding.

Cordycepin protects against β-amyloid and ibotenic acid-induced hippocampal CA1 pyramidal neuronal hyperactivity

Cordycepin exerts neuroprotective effects against excitotoxic neuronal death. However, its direct electrophysiological evidence in Alzheimer's disease (AD) remains unclear. This study aimed to explore the electrophysiological mechanisms underlying the protective effect of cordycepin against the excitotoxic neuronal insult in AD using whole-cell patch clamp techniques. β-Amyloid (Aβ) and ibotenic acid (IBO)-induced injury model in cultured hippocampal neurons was used for the purpose. The results revealed that cordycepin significantly delayed Aβ + IBO-induced excessive neuronal membrane depolarization. It increased the onset time/latency, extended the duration, and reduced the slope in both slow and rapid depolarization. Additionally, cordycepin reversed the neuronal hyperactivity in Aβ + IBO-induced evoked action potential (AP) firing, including increase in repetitive firing frequency, shortening of evoked AP latency, decrease in the amplitude of fast afterhyperpolarization, and increase in membrane depolarization. Further, the suppressive effect of cordycepin against Aβ + IBO-induced excessive neuronal membrane depolarization and neuronal hyperactivity was blocked by DPCPX (8-cyclopentyl-1,3-dipropylxanthine, an adenosine A₁ receptor-specific blocker). Collectively, these results revealed the suppressive effect of cordycepin against the Aβ + IBO-induced excitotoxic neuronal insult by attenuating excessive neuronal activity and membrane depolarization, and the mechanism through the activation of A₁R is strongly recommended, thus highlighting the therapeutic potential of cordycepin in AD.

Role of adenosine A1 receptor within nucleus tractus solitarii in dexmedetomidine-induced increase in baroreflex sensitivity in rats / 中华麻醉学杂志

Objective To evaluate the role of A1 adenosine receptor ( A1 AR) within the nucleus tractus solitarii ( NTS ) in dexmedetomidine-induced increase in baroreflex sensitivity ( BRS ) in rats. Methods Thirty-two clean-grade healthy male Sprague-Dawley rats, weighing 240-280 g, were divided in-to 4 groups ( n=8 each) using a random number table method: control group ( group C) , solvent control group ( group S) , dexmedetomidine group ( group D) , and dexmedetomidine plus 8-cyclopentyl-1,3-diprop-ylxanthine (DPCPX, a highly selective A1AR blocker) group (group DD). After the rats were anesthe-tized, 1 μl drug liquid was injected into the right NTS with a brain stereotaxic apparatus. Oneμl normal sa-line was injected into the right NTS in C and D groups, 1 μl dimethyl sulfoxide in group S, and 1 μl DPCPX in group DD. After catheters were implanted into the femoral vein, dexmedetomidine was intrave-nously infused as a bolus of 100μg/kg over 15 min followed by an infusion of 50μg·kg-1 ·h-1 for 105 min in D and DD groups. The equal volume of normal saline was given instead of dexmedetomidine in C and S groups. BRS was measured using phenylephrine immediately before intravenous infusion (T0) and at 60 and 120 min after beginning of infusion ( T1,2 ) . Results Compared with C and S groups, the BRS was signifi-cantly increased at T1,2 in D and DD groups ( P＜0. 05) . Compared with group D, the BRS was significantly decreased at T1,2 in group DD ( P＜0. 05) . Conclusion A1 AR within the NTS is involved in dexmedetomi-dine-induced increase in BRS in rats.

The Advance of Escitalopram's Drug-induced QTc Prolongation / 中国药学杂志

Escitalopram, a selective serotonin re-uptake inhibitor (SSRI) antidepressant which is the (S)-enantiomer of citalopram, is worldwide used for the treatment of depressive and anxious disorders in clinical practice, however, recent data have indicated that high therapeutic escitalopram doses may cause the potential of QTc prolongation effect, which is a predisposing factor for arrhythmia. Nevertheless, in March 2012, the Food and Drug Administration (FDA) issued a safety bulletin advising the daily dosage of escitalopram should be restricted to a maximum of 20 mg daily in healthy adults and 10 mg maximum in high risk patients (eg>60 years of age). In this review, we aimed to investigate what factors can affect and how escitalopram gives rise to QTc prolongation.

Involvement of adenosine A1 receptor in electroacupuncture-mediated inhibition of astrocyte activation during neuropathic pain / Envolvimento do receptor de adenosina A1 na inibição da ativação de astrócitos mediada por eletroacupuntura durante a dor neuropática

ABSTRACT Neuropathic pain is a chronic pain condition caused by damage or dysfunction of the central or peripheral nervous system. Electroacupuncture (EA) has an antinociceptive effect on neuropathic pain, which is partially due to inhibiting astrocyte activation in the spinal cord. We found that an intrathecal injection of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective adenosine A1 receptor antagonist, reversed the antinociceptive effects of EA in a chronic constriction injury-induced neuropathic pain model. The expression of GFAP in L4-L6 spinal cord was significantly upgraded, while DPCPX suppressed the effect of the EA-mediating inhibition of astrocyte activation, as well as wiping out the EA-induced suppression of cytokine content (TNF-α). These results indicated that the adenosine A1 receptor is involved in EA actions during neuropathic pain through suppressing astrocyte activation as well as TNF-α upregulation of EA, giving enlightenment to the mechanisms of acupuncture analgesia and development of therapeutic targets for neuropathic pain.

RESUMO A dor neuropática é uma condição de dor crônica causada por dano ou disfunção do sistema nervoso central ou periférico. A eletroacupuntura (EA) tem um efeito antinociceptivo durante a dor neuropática, que é parcialmente devido à inibição da ativação de astrócitos na medula espinhal. Descobrimos que a injeção intratecal de 8-ciclopentil-1,3-dipropilxantina (DPCPX), um antagonista seletivo do receptor de adenosina A1, reverteu os efeitos antinociceptivos da EA no modelo de dor neuropática induzida por lesão por constrição crônica (CCI). A expressão da GFAP na medula espinal L4-L6 foi significativamente melhorada, enquanto a DPCPX suprimiu o efeito da inibição mediadora da EA na ativação de astrócitos, bem como eliminou a supressão induzida pela EA do conteúdo de citocina (TNF-α). Esses resultados indicam que o receptor de adenosina A1 está envolvido nas ações da EA durante a dor neuropática, suprimindo a ativação astrocitária, bem como o aumento da TNF-α na EA, fornecendo esclarecimentos sobre os mecanismos de analgesia da acupuntura e o desenvolvimento de alvos terapêuticos para dor neuropática.

Regulation of Matrix Metalloproteinase 2 Expression by an Adenosine A1 Agonist in Trabecular Meshwork Cells

PURPOSE: We investigated the extent of adenosine A1 agonist-induced expression and regulation of matrix metalloproteinase 2 (MMP-2) synthesis in human trabecular meshwork cells (HTMC). METHODS: Primary HTMC cultures were exposed to 0.1 or 1.0 µM N6-cyclohexyladenosine (CHA) for 2 h in the presence or absence of an inhibitor thereof, 8-cyclopentyl-1,3-dimethylxanthine (CPT). The expression level of mRNA encoding MMP-2 was assessed via reverse transcription-polymerase chain reaction, and the levels of tissue inhibitor of metalloproteinase 2 (TIMP2) and membrane-type-1 MMP (MT1-MMP) measured by Western blotting. The permeability of the HTMC monolayer was assessed with the aid of carboxyfluorescein. RESULTS: CHA at 1.0 µM increased the permeability of the HTMC monolayer (p = 0.003) and CHA at both 0.1 and 1.0 µM significantly increased MMP-2 mRNA expression, which was inhibited by co-exposure to CPT (all p < 0.05). CHA increased MMP-2 activity, decreased that of TIMP2, and increased that of MT1-MMP (all p < 0.05). CONCLUSIONS: CHA increased the permeability of the HTMC monolayer and increased MMP-2 activity, decreased TIMP2 activity, and increased MT1-MMP activity. Thus, regulation of TIMP2 and MT1-MMP expression may be involved in the adenosine A1 agonist-induced increase in MMP-2 activity.

Effects of adenosine receptor agonist on the rocuronium-induced neuromuscular block and sugammadex-induced recovery / 대한마취과학회지

BACKGROUND: Several types of receptors are found at neuromuscular presynaptic membranes. Presynaptic inhibitory A1 and facilitatory A2A receptors mediate different modulatory functions on acetylcholine release. This study investigated whether adenosine A1 receptor agonist contributes to the first twitch tension (T1) of train-of-four (TOF) stimulation depression and TOF fade during rocuronium-induced neuromuscular blockade, and sugammadex-induced recovery. METHODS: Phrenic nerve-diaphragm tissues were obtained from 30 adult Sprague-Dawley rats. Each tissue specimen was randomly allocated to either control group or 2-chloroadenosine (CADO, 10 μM) group. One hour of reaction time was allowed before initiating main experimental data collection. Loading and boost doses of rocuronium were sequentially administered until > 95% depression of the T1 was achieved. After confirming that there was no T1 twitch tension response, 15 min of resting time was allowed, after which sugammadex was administered. Recovery profiles (T1, TOF ratio [TOFR], and recovery index) were collected for 1 h and compared between groups. RESULTS: There were statistically significant differences on amount of rocuronium (actually used during experiment), TOFR changes during concentration-response of rocuronium (P = 0.04), and recovery profiles (P < 0.01) of CADO group comparing with the control group. However, at the initial phase of this experiment, dose-response of rocuronium in each group demonstrated no statistically significant differences (P = 0.12). CONCLUSIONS: The adenosine A1 receptor agonist (CADO) influenced the TOFR and the recovery profile. After activating adenosine receptor, sugammadex-induced recovery from rocuronium-induced neuromuscular block was delayed.

Adonesine A1 receptor and megalin defect in diabetic mice with early kidney disease / 中华肾脏病杂志

Objective To observe the effect of adenosine A1 receptor (A1AR) on the megalin defect in type 1 diabetic mice with early kidney disease.Methods 7-8 week-old,baseline body weight and fasting blood glucose matched wild type (WT) C57BL/6J mice were selected,and randomly divided into two groups:control group (n=6) and WT DM group (n=6).In the same way,male A1AR knock-out C57BL/6J mice were selected as A1AR-/-DM group (n=6).DM model was established by intraperitoneal injection of streptozocin.The blood glucose (BG),body weight (BW),kidney weight (KW),24 h proteinuria (24hUP) and albumin creatine ratio (ACR) were measured at 4 weeks.The renal pathological lesion was observed and the expression of megalin in proximal tubules was examined by immunohistochemistry.The expression of caspase-1,IL-18 and A1AR were detected by Western blotting.Results At 4th week,compared with WT control mice,the BG,BW,KW and 24hUP of WT DM mice were increased significantly (n=6,P ＜ 0.01),with the pathological glomerular enlargement,mesangial cell proliferation,extracellular matrix accumulation and renal tubule hypertrophy being observed.Immunohistochemistry revealed decreased expression of megalin,an important multiligand protein receptor on the brush border of proximal tubular epithelial cells in WT DM mice,which was correlated with 24hUP (r=-0.645,P ＜ 0.01).Compared with the control mice,the expressions of caspase-1,IL-18 and A1AR were significantly increased in WT DM mice (P ＜ 0.05).For A1AR-/-DM mice,more serious pathological lesion and megalin defect,together with increasing of casapase-1 and heavier proteinuria were observed than those in WT DM mice.Conclusion A1AR may play a protective role in megalin expression of diabetic mice with early kidney disease,in which the mechanism may be associated with caspase-1 related pyroptosis pathway.The details need further exploration.

Effect of Electroacupuncture on the Expression of Adenosine A1Receptor in Hypothalamus and Spinal Cord of Adjuvant Arthritis Rats / 上海针灸杂志

Objective To observe the effect of adenosine A1 receptor on pain and electroacupuncture analgesia, and to explore the action mechanism of electroacupuncture in analgesia.Method Adjuvant arthritis rats were taken as the study subjects. 24 rats were randomized into a normal group, a model group and an electroacupuncture (EA) group, 8 rats in each group. The pain threshold was evaluated by using thermal radiation method, immunohistochemical method and real-time fluorescence quantitative PCR were adopted to observe the expression of adenosine A1 receptor in hypothalamus and spinal cord.Result One day after modeling, the pain thresholds of right hind paw in the model group and EA group were significantly changed compared to that before modeling in the same group (P<0.01). The pain thresholds of right hind paw in the model group and EA group were significantly different from that in the normal group one day after modeling (P<0.01). 7 d after modeling, the pain threshold of right hind paw in the model group was still significantly lower than that before modeling in the same group (P<0.01), and it was significantly different from that in the normal group and EA group (P<0.01). The pain threshold was significantly enhanced in the EA group 7 d after modeling, and was significantly different from that of 1 d after modeling in the same group (P<0.01). The positive cell expression was lower in the model group and was significantly different from that in the normal group (P<0.01). The expression of adenosine A1 receptor in the EA group was markedly higher than that in the model group (P<0.01). The expression of adenosine A1 receptor in hypothalamus and spinal cord of the model group was significantly lower than that of the normal group (P<0.01). The expression of adenosine A1 receptor in hypothalamus and spinal cord of EA group was markedly higher than that of the model group (P<0.01).Conclusion EA can up-regulate the expression of adenosine A1 receptor in hypothalamus and spinal cord of adjuvant arthritis rats.

Role of adenosine A1 receptors in hippocampal neurons in cognitive dysfunction caused by isoflurane anesthesia in aged mice / 中华麻醉学杂志

Objective To evaluate the role of adenosine A1 receptors in hippocampal neurons in the cognitive dysfunction caused by isoflurane anesthesia in aged mice.Methods Sixteen male adenosine A1 receptor gene knockout homozygote mice (gene knockout mice) and 16 male wild-type mice,aged 18-22 months,weighing 27-32 g,were studied.Each type of mice was randomly divided into 2 groups (n=8 each) using a random number table:control group (group C) and isoflurane anesthesia group (group Ⅰ).Mice inhaled 1.4％ isoflurane in 100％ O2 for 2 h in group Ⅰ,and 100％ O2 for 2 h in group C.All the mice underwent Morris water maze test at 24 h after isoflurane or O2 inhalation.After the test,the mice were sacrificed and the hippocampal tissues were harvested to determine the number of β-amyloid1-42 (Aβ1-42) plaques (using immunohistochemistry) and expression of phosphorylated tau (p-tau) protein,and 2B subunit-containing N-methyl-D-aspartate receptors (NR2B) (by Western blot analysis).Results Compared with group C of wild type mice,the escape latency was significantly prolonged,the number of Aβ1-42 plaques was enlarged,the expression of p-tau protein was up-regulated,and the expression of N R2B was down-regulated in group Ⅰ of wild type mice.Compared with group Ⅰ of wild type mice,the escape latency was significantly shortened,the number of Aβ1-42 plaques was decreased,the expression of p-tau protein was down-regulated,and the expression of NR2B was up-regulated in group Ⅰ of gene knockout mice.There was no significant difference in the parameters mentioned above between group Ⅰ and group C of gene knockout mice.Conclusion Adenosine A1 receptors in hippocampal neurons mediate isoflurane anesthesia-induced cognitive dysfunction in aged mice,and the mechanism may be related to promotion of deposition of Aβ,phosphorylation of tau protein and inhibition of activities of NR2B.

Proliferation and activation of acetaldehyde-induced HSC-T6 cells through RNA inference targeting adenosine A1 and A2 A receptors / 中国药理学通报

Aim To investigate the influence of down-regulating adenosine A1 receptor and adenosine A2 A receptor gene expression on proliferation and activation of acetaldehyde-induced hepatic stellate cell-T6 cells through siRNA. Methods Alcoholic liver fibrosis in vitro model was constructed by inducing HSC-T6 cells with acetaldehyde. siRNA targeting A1R and A2AR were designed and synthesized according to its mRNA. The siRNA was transfected into rat HSC-T6 cells by li-posome LipofectamineTM 2000. HSC cell proliferation was measured by MTT. The mRNA levels of A1R, A2AR, α-SMA, Collagen I in the supernatant of the cell culture were measured by Quantitative Real-Time PCR. The protein levels of A1R, A2AR, α-SMA, Collagen I were measured by Western blot. Results A1 R and A2 AR siRNA effectively inhibited the cell proliferation, and they also significantly decreased the levels of A1R, A2AR,α-SMA, Collagen I, suggesting that A1 R and A2 AR might be potential target genes in the alcoholic liver fibrosis. Conclusions Silencing A1 R or A2 AR by RNAi can significantly inhibit the HSC proliferation, A1R and A2AR may be potential therapeutic target genes for alcoholic liver fibrosis.

Effects of Adenosine and the Selective Adenosine Receptor Agonist on the Expression of Interleukin-18 in Patient with Unstable Angina / 天津医药

Objective To observe the effects of adenosine and adenosine receptor agonist on the expression of inter?leukin-18 (IL-18) in patients with unstable angina (UAP), and the mechanism of adenosine receptor agonists thereof. Meth?ods Fifteen UAP and 15 healthy volunteers were included in this study. The effects of adenosine and the selective adenos?ine receptor agonists on the expression of IL-18 were measured by enzyme-linked immunosorbent assay (ELISA). Results The lower concentration of adenosine (1-100μmol/L) increased the expression of IL-18 in UAP group;whereas the higher concentration of adenosine (1 mmol/L) inhibited the expression of IL-18. The adenosine A1 receptor agonist and A3 receptor agonist increased the expression of IL-18, while the adenosine A2a receptor agonist inhibited the expression of IL-18. There was no significant effect for A2b receptor agonist on the expression of IL-18. Conclusion The lower concentrations of ade?nosine can enhance the expression of IL-18 through adenosine A1 and A3 receptors in UAP. The higher concentrations of ade?nosine can inhibit the expression of IL-18 through adenosine A2a receptors. Adenosine can promote IL-18 expression in lower concentrations.

Molecular mechanisms of ischemic preconditioning mediated neuroprotection / 国际脑血管病杂志

A large number of animal experiments have confirmed that ischemic preconditioning can produce a powerful organ protective effect,but the progress and results of the conversion of animal experiments to clinical trials are unsatisfactory.It has great significance for studying of molecular mechanisms of ischemic preconditioning mediated neuroprotection,searching for safe and effective preconditioning induced ways which can be converted to clinical practice,improving the tolerance of nerve tissue ischemia and hypoxia in stroke and surgical patients and achieving a safe and effective neuroprotection.This article reviews the molecular mechanisms of ischemic preconditioning mediated neuroprotection from the aspects of pretreatment of activated receptor,mitochondria,transcription factor,and protein kinase.

Metallothionein mediates cardioprotection induced by delayed preconditioning with adenosine A1 receptor agonist in rabbits / 中南大学学报(医学版)

Objective To investigate the protective effect of adenosine A1 receptor agonist (2-chloro-N6-cyclopentyladenosine, CCPA) delayed preconditioning on myocardial ischemia reperfu-sion injury and the potential mechanism in rabbits. Methods Thirty New Zealand male white rabbits were randomly assigned to 3 groups:a control group, an I/R group, and a CCPA group. CCPA group was given CCPA 0.1 mg/kg before the myocardial ischemia. Twenty-four hours later I/R group and CCPA group underwent 40 min of coronary occlusion followed reperfusion for 2 h. At the end of the reperfusion, blood samples were taken from the arterial line for determining the plasma level of malondialdhyde and superoxide dismutase activity. The infarct size and area at risk were de-fined by Evans and TIC staining. The heart was harvested and levels of metallothionein (MT) were determined by Western blot, and ultrastructures were observed under the electron microscope. Results The MT level of CCPA group was significantly higher than that of the I/R group (P<0.05). CCPA significantly reduced the infarct size (22.1%±3.8% in the CCPA group) of the left yen-tricular area at risk as compared with the control (41.8%±4.3% in the I/R group,P<0.05). The injury of I/R group was worse than that of the CCPA group under the light microscope. CCPA group had higher superoxide dismutase and lower malondialdhyde than those of the I/R group. Con-clusion CCPA can increase the level of metallothionein during ischemia-reperfusion, which may be part of the molecular mechanism of CCPA delayed preconditioning on cardioprotection.

The dynamic changes of adenosine receptors imbalance during kindling by lithium chloride-pilocarpine / 中华神经科杂志

Objective To investigate the dynamic changes of adenosine receptors, A1 (A1R) and A2a (A2aR) in the brain from the acute to chronic phase after kindling and to explore the correlation between seizure and expression level of A1R and A2aR. MethodsRats were randomly selected into the testing model, reference and normal control groups.Testing rats were kindled by lithium choride-pilocarpine, reference rats were treated with saline, and no treatment was given in normal control group. The dynamic expression of A1R and A2aR were detected by RT-PCR, immunofluorescence staining and Western blot at time-points of 24 hours, 1 month and 6 months post-kindling. Results In the acute phase of 24 hours after kindling, the A1R expression level (mRNA level was (1. 1483 ±0. 1182); Western blot result was ( 0. 7872± 0. 0621 ) ; immunofluorescence staining count was ( 76. 17 ± 4. 62 )/HP) was increased and A2aR (mRNA level was (0. 8338±0. 0572) ; Western blot result was (0. 2098 ±0. 0257) ; immunofluorescence staining count was (43. 83 ± 5. 12 )/HP) was decreased.The results showed statistically difference compared with the reference and normal groups (P< 0. 05 ). In the later chronic phase of 1 month and 6 months after kindling, the expression level of A1R was decreased and A2aR was increased. These data revealed statistically significant difference (P <0. 01 ). Furthermore, the comparison of the results in 1 month and 6 months after kindling found that the expression of AIR was lower in 6 months (mRNA level was (0. 5682 ±0. 0443) ; Western blot result was (0. 7749 ±0. 0262) ; immunofluorescence staining count was (38. 50 ±4. 81 )/HP) than in 1 month and that of A2aR was higher in 6 months (mRNA level was (1. 2169±0. 0332) ; Western blot result was (0. 7080 ±0. 0371 ); immunofluorescence staining count was (114. 50 ± 4. 04)/HP). The differences were statistical significant (t = - 19. 02--13.28, P < 0. 05). ConclusionsThe expressions of A1R and A2AR during and after kindling presents a bidirectional change. In the acute phas the expression of AR is regulated to suppress seizures. While in the chronic phase, the repeated seizures result in the change of A1R and A2aR expression in the opposite direction. This mechanism plays an important role in refractory seizures.

Protective effect of adenosine in diabetic neuropathic pain is mediated through adenosine A1-receptors.

Diabetic neuropathic pain is generally considered to be one of the most troublesome complications affecting diabetic patients and current therapy provides inadequate pain relief. In the present study, the effect of adenosine was investigated in a model of diabetic neuropathic pain. Diabetes was induced by streptozotocin (65 mg/kg, ip) in male Sprague Dawley rats and subjected to thermal (cold and hot) and chemical (formalin) stimuli. Diabetic rats developed hyperalgesia by the end of six weeks in thermal and chemical stimuli test. Adenosine (100, 200 and 500 mg/kg, ip) produced significant reversal of responses to thermal and chemical stimuli in diabetic rats. 8-Cyclopentyl-1, 3-dipropylxanthine (DPCPX 1 mg/kg, ip), an adenosine A1-receptor antagonist, but not 3,7-dimethyl-l-propargylxanthine (DMPX 1 mg/kg, ip), an adenosine A2A-receptor antagonist, reversed the protective effect of adenosine. These results indicate that adenosine is an effective analgesics in a model of diabetic neuropathy, and the protection produced by adenosine is via stimulation of adenosine A1-receptors.