BackgroundThe cholinergic anti-inflammatory pathway (CAP) primarily functions through acetylcholine (ACh)-alpha7 nicotinic acetylcholine receptor (α7nAChR) interaction on macrophages to control peripheral inflammation. Interestingly, ACh can also bind α7nAChRs on microglia resulting in neuroprotective effects. However, ACh effects on astrocytes remain elusive. Here, we investigated the effects of nicotine, an ACh receptor agonist, on the cytokine and cholinesterase production of immunocompetent human astrocytes stimulated with interleukin 1β (IL-1β) in vitro. In addition, the potential involvement of prostaglandins as mediators of nicotine was studied using cyclooxygenase 2 (COX-2) inhibition.MethodsCultured human fetal astrocytes were stimulated with human recombinant IL-1β and treated simultaneously with nicotine at different concentrations (1, 10, and 100 μM). Cell supernatants were collected for cytokine and cholinesterase profiling using ELISA and MesoScale multiplex assay. α7nAChR expression on activated human astrocytes was studied using immunofluorescence. For the COX-2 inhibition studies, enzyme activity was inhibited using NS-398. One-way ANOVA was used to perform statistical analyses.ResultsNicotine treatment dose dependently limits the production of critical proinflammatory cytokines such as IL-6 (60.5 ± 3.3, %inhibition), IL-1β (42.4 ± 1.7, %inhibition), and TNF-α (68.9 ± 7.7, %inhibition) by activated human astrocytes. Interestingly, it also inhibits IL-8 chemokine (31.4 ± 8.5, %inhibition), IL-13 (34.243 ± 4.9, %inhibition), and butyrylcholinesterase (20.8 ± 2.8, %inhibition) production at 100 μM. Expression of α7nAChR was detected on the activated human astrocytes. Importantly, nicotine’s inhibitory effect on IL-6 production was reversed with the specific COX-2 inhibitor NS-398.ConclusionsActivation of the cholinergic system through α7nAChR agonists has been known to suppress inflammation both in the CNS and periphery. In the CNS, earlier experimental data shows that cholinergic activation through nicotine inhibits microglial activation and proinflammatory cytokine release. Here, we report similar anti-inflammatory effects of cholinergic activation on human astrocytes, at least partly mediated through the COX-2 pathway. These results confirm the potential for cholinergic neuroprotection, which is looked upon as a promising therapy for neuroinflammation as well as neurodegenerative diseases and stroke. Our data implicates an important role for the prostaglandin system in cholinergic regulatory effects.
Altered activity of the proteolytic machine-the 26S proteasome is observed in many disease conditions. Hence, either inhibition or activation of the 26S proteasome is thought to be a novel therapy for treatment of certain diseases such as cancer and neurodegenerative disorders. In this study, we tested the potential of cinnamon and one of its active ingredients, procyanidin-B2 (PCB2), in inhibiting the catalytic activities of the proteasome and suppressing prostate cancer cell growth. Proteasome activities were measured using fluorogenic substrates specific for the different enzymatic activities of the 26S proteasome by flourometry. Cell viability was assessed using the 3-[4, 5-dimethylthiazol-2-yl]-2.5-diphenyl-tetrazolium bromide assay, while apoptosis was examined by Hoechst and propidium iodide staining and caspase-3 activity. Both, the cinnamon extract and its PCB2-enriched F2 fraction inhibited the catalytic activities of the purified proteasome and the proteasome in cancer cells but not in normal cells. Furthermore, cinnamon and its active component decreased cell proliferation of human prostate cancer cells but not normal lung cells, decreased expression of anti-apoptotic and angiogenic markers in prostate cancer cell lysates. These results demonstrate that cinnamon extract and its PCB2-enriched fraction act as proteasome inhibitors and have prospects as anti-cancer agents. © 2018 IUBMB Life, 70(5):445-457, 2018.
The enantiopure synthesis of clavatustides A (1) and B (3) were accomplished by seven step synthetic protocol starting from commercially available (R)-Phenyllactic acid. As the optical rotation values of synthetic (R)-clavatustides A and B were not in agreement with the reported values, the corresponding antipodes 2 and 4 were synthesized from (S)-Phenyllactic acid to address the issue. Both (R) and (S) clavatustides A and B were evaluated for their antiproliferative activity against three human cancer cell lines using MTT assay.The cervical cancer cell line (HeLa) was found to be most sensitive to the test compounds in decreasing the cell viability. S-isomers (2 and 4) were found to exhibit better anti-proliferative activity when compared to its enantiomers (R-isomers) with IC 50s 24.5 and 26.8 µM respectively against HeLa cell lines. All the compounds tested had a minimal effect on the cell viability of the normal lung cells, indicating that these compounds are not toxic to normal cells. (R)-3-Phenyl-Lac-O t Bu (13): TLC (Hexane: EtOAc = 9 : 1); [α] D 25 = +9.20 (c 1.0, CHCl 3 ); SFC: Chiralpak AD-H(4.6x250) mm5µ, CO 2 -0.5 % DEA in MeOH, 90:10 flow rate = amino)acetamido)benzamido)benzoate (11): To a stirred solution of N-Boc-N-ethyl Gly (0.529 g, 2.60 mmol) in THF were added DIPEA (1.43 mL, 8.66 mmol), HATU (1.23 g, 3.26 mmol) at 0 o C and stirred for 30 min. A solution of compound 17 (1.0 g, 2.17 mmol) in THF (15 mL) was added to the reaction mixture at 0 °C and stirred for 48 h at room temperature. The reaction mixture was quenched with cold water (25 mL) and extracted with EtOAc (3 × 25 mL).The combined organic layer were washed with water, brine (25 mL), dried over anhydrous
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