Capsaicin inhibits the production of tumor necrosis factor alpha by LPS-stimulated murine macrophages, RAW 264.7: a PPARgamma ligand-like action as a novel mechanism

Capsaicin, a major ingredient of hot pepper, is considered to exhibit anti-inflammatory properties. Our previous study demonstrated that capsaicin inhibited the production of pro-inflammatory mediators through NF-kappaB inactivation in LPS-stimulated macrophages. In order to further clarify the mechanism underlying the anti-inflammatory action of capsaicin, we investigated whether capsaicin alters PPARgamma activity, which regulates the production of the pro-inflammatory cytokine TNFalpha. Capsaicin significantly inhibited the production of TNFalpha by macrophages in a dose-dependent manner. Simultaneous exposure of the cells to capsaicin and PPARgamma agonist troglitazone or RXR agonist LG100268 resulted in stronger inhibition of TNFalpha production compared to the cells treated with either capsaicin, troglitazone, or LG100268 alone. Luciferase reporter assay revealed that capsaicin induced GAL4/PPARgamma chimera and full length PPARgamma (PPRE) transactivations in a dose-dependent manner. Furthermore, a specific PPARgamma antagonist T0070907 abrogated the inhibitory action of capsaicin on LPS-induced TNFalpha production by RAW 264.7 cells, indicating that capsaicin acts like a ligand for PPARgamma. Our data demonstrate for the first time that the anti-inflammatory action of capsaicin may be mediated by PPARgamma activation in LPS-stimulated RAW 264.7 cells.     

Capsaicin Interaction with TRPV1 Channels in a Lipid Bilayer: Molecular Dynamics Simulation

Transient receptor potential vanilloid subtype 1 (TRPV1) is a heat-sensitive ion channel also involved in pain sensation, and is the receptor for capsaicin, the active ingredient of hot chili peppers. The recent structures of TRPV1 revealed putative ligand density within the S1 to S4 voltage-sensor-like domain of the protein. However, questions remain regarding the dynamic role of the lipid bilayer in ligand binding to TRPV1. Molecular dynamics simulations were used to explore behavior of capsaicin in a 1-palmitoyl-2-oleoyl phosphatidylcholine bilayer and with the target S1–S4 transmembrane helices of TRPV1. Equilibrium simulations reveal a preferred interfacial localization for capsaicin. We also observed a capsaicin molecule flipping from the extracellular to the intracellular leaflet, and subsequently able to access the intracellular TRPV1 binding site. Calculation of the potential of mean force (i.e., free energy profile) of capsaicin along the bilayer normal confirms that it prefers an interfacial localization. The free energy profile indicates that there is a nontrivial but surmountable barrier to the flipping of capsaicin between opposing leaflets of the bilayer. Molecular dynamics of the S1–S4 transmembrane helices of the TRPV1 in a lipid bilayer confirm that Y511, known to be crucial to capsaicin binding, has a distribution along the bilayer normal similar to that of the aromatic group of capsaicin. Simulations were conducted of the TRPV1 S1–S4 transmembrane helices in the presence of capsaicin placed in the aqueous phase, in the lipid, or docked to the protein. No stable interaction between ligand and protein was seen for simulations initiated with capsaicin in the bilayer. However, interactions were seen between TRPV1 and capsaicin starting from the cytosolic aqueous phase, and capsaicin remained stable in the majority of simulations from the docked pose. We discuss the significance of capsaicin flipping from the extracellular to the intracellular leaflet and mechanisms of binding site access by capsaicin.     

Hypolipidemic and antioxidant effects of curcumin and capsaicin in high-fat-fed rats

The beneficial hypolipidemic and antioxidant influences of the dietary spice compounds curcumin and capsaicin were evaluated. Curcumin, capsaicin, or their combination were included in the diet of high-(30%)-fat-fed rats for 8 weeks. Dietary high-fat-induced hypertriglyceridemia was countered by dietary curcumin, capsaicin, or their combination by 12%-20%. Curcumin, capsaicin, and their combination also produced a slight decrease in serum total cholesterol in these animals. Serum alpha-tocopherol content was increased by dietary curcumin, capsaicin, and their combination in high-fat-fed rats. Serum total thiol content in high-fat-fed animals and serum ascorbic acid in normal animals was elevated by the combination of curcumin and capsaicin. Hepatic glutathione was increased by curcumin, capsaicin, or their combination in normal animals. Hepatic glutathione and alpha-tocopherol were increased, whereas lipid peroxide level was reduced by dietary curcumin and combination of curcumin and capsaicin in high-fat-fed animals. Serum glutathione peroxidase and glutathione transferase in high-fat-fed rats were generally higher as a result of dietary curcumin, capsaicin, and the combination of curcumin and capsaicin. Hepatic glutathione reductase and glutathione peroxidase were significantly elevated by dietary spice principles in high-fat-fed animals. The additive effect of the 2 bioactive compounds was generally not evident with respect to hypolipidemic or antioxidant potential. However, the effectiveness of the combination was higher in a few instances.     

Capsaicin-induced activation of erythrocyte membrane sodium/potassium and calcium adenosine triphosphatases

Capsaicin is the pungent ingredient present in hot peppers of the genus Capsicum. Capsaicin's effect on sensory neurons has been well studied; however, its effect on non-neuronal cells is still not fully understood. This study was undertaken to evaluate the effect of capsaicin on erythrocyte membrane enzymes: Na+/K(+)-ATPase and Ca(2+)-ATPase. Treatment with capsaicin (0.01-100 microM) caused a transient increase in the activities of both enzymes; the effect declined at lower concentrations of capsaicin, and no significant effect was observed at 0.01 microM capsaicin. The effect of capsaicin was fast with a significant (p<0.01) activation of enzyme activity observed within minutes of incubation. The findings on the effect of capsaicin on human erythrocyte membrane enzymes Na+/K(+)-ATPase and Ca(2+)-ATPase signify the importance of the non-neuronal effects of capsaicin, and the need for evaluating the physiological impact of high capsaicin (capsicum) consumption in some regions of the world.     

Capsaicin inhibits platelet-activating factor-induced cytosolic Ca2+ rise and superoxide production

Platelet-activating factor (PAF) is an important participant in the inflammatory process. We studied the regulation of PAF activity by capsaicin in human promyelocytic leukemia HL-60 cells. Capsaicin inhibited PAF-induced superoxide production in a concentration-dependent manner. In addition to PAF, the fMLP- and extracellular ATP-induced superoxide productions were inhibited by capsaicin, whereas PMA-induced superoxide production was not affected. In the PAF-stimulated cytosolic Ca2+ increase, capsaicin inhibited in particular the sustained portion of the raised Ca2+ level without attenuation of the peak height. In the absence of extracellular Ca2+, the PAF-induced Ca2+ elevation was not inhibited by capsaicin because capsaicin only inhibited the Ca2+ influx from the extracellular space. In addition, capsaicin did not affect PAF-induced inositol 1,4,5-trisphosphate production, suggesting that phospholipase C activation by PAF is not affected by capsaicin. Store-operated Ca2+ entry (SOCE) induced by thapsigargin was inhibited by capsaicin in a concentration-dependent manner. This capsaicin effect was also observed on thapsigargin-induced Ba2+ and Mn2+ influx. Furthermore, capsaicin's inhibitory effect on the thapsigargin-induced Ca2+ rise overlapped with that of SK&F96365, an inhibitor of SOCE. Both capsaicin and SK&F96365 also inhibited PAF-induced cytosolic superoxide generation in HL-60 cells differentiated by all-trans-retinoic acid. Our data suggest that capsaicin exerts its anti-inflammatory effect by inhibiting SOCE elicited via PLC activation, which occurs upon PAF activation and results in the subsequent superoxide production.     

Capsaicin-induced beta-adrenergic action on energy metabolism in rats: influence of capsaicin on oxygen consumption, the respiratory quotient, and substrate utilization

The mode of action of capsaicin on energy metabolism was investigated in rats. The oxygen consumption was higher when capsaicin (6.0 mg/kg) was intraperitoneally injected than when it was not injected. The respiratory quotient (R.Q.) increased and then decreased after the administration of capsaicin. The levels of serum glucose and immunoreactive insulin rapidly increased after the administration of capsaicin. Also, liver glycogen rapidly decreased, in contrast to the serum glucose concentration which rapidly increased. The serum-free fatty acid level gradually increased after the administration of capsaicin. These alterations in energy metabolism on the administration of capsaicin were similar to those in the metabolism of epinephrine, and were specifically inhibited by various beta-adrenergic blockers. On the other hand, the alterations were not affected by pretreatment with alpha-adrenergic or ganglion blockers. These results suggest that the mode of action of capsaicin on the enhancement of energy metabolism in rats comprises a direct (as an agonist) and/or an indirect (via catecholamine) beta-adrenergic action. Therefore, it was speculated that the adrenergic action of capsaicin resulted, at least in part, in a decrease in the perirenal adipose tissue weight and serum triglyceride concentration in rats fed a high fat diet supplemented with capsaicin (T. Kawada et al., J Nutr 116:1272-1278, 1986).     

Repetitive intradermal capsaicin: differential effect on pain and areas of allodynia and punctate hyperalgesia

This study examined the effect of repeated intradermal capsaicin injections on capsaicin pain intensity and areas of allodynia and punctate hyperalgesia. Seventeen healthy volunteers participated in four sessions separated by at least 5 days. Each session included four intradermal injections of 10 microg of capsaicin. In one session injections were given with 0.5 cm and 6 min intervals, in a second with 0.5 cm and 15 min intervals, in a third with 0.5 cm and 24 min intervals, and in a fourth session with 4 cm and 15 min intervals. Following each injection capsaicin pain intensity was measured in the first 5 min, the area of allodynia at 5 min and area of punctate hyperalgesia at 10 min. With 6 min and 0.5 cm between repeated injections, capsaicin pain intensity decreased significantly whereas areas of allodynia and punctate hyperalgesia increased. In contrast, both capsaicin pain intensity and areas of allodynia and punctate hyperalgesia increased when the interval between injections was 24 min and 0.5 cm or 15 min and 4 cm. With 15 min and 0.5 cm between injections, capsaicin pain intensity did not change, whereas areas of allodynia and punctate hyperalgesia increased. There were no significant relations between capsaicin pain intensity and areas of allodynia and punctate hyperalgesia after first injections. The findings indicate that the response to intradermal injection of capsaicin is dependent on the time and distance between injections. The lack of significant relation between capsaicin pain intensity and area of allodynia and punctate hyperalgesia suggests that the two phenomena are mediated by different central mechanisms.     

Involvement of a capsaicin-sensitive TRPV1-independent mechanism in lipopolysaccharide-induced fever in chickens

It has been demonstrated that capsaicin blocks lipopolysaccharide (LPS)-induced fever in mammals. In this study, we investigated TRPV1 (transient receptor potential ion channel of vanilloid subtype-1)-independent action of capsaicin on LPS-induced fever in chickens. The chicken is a valuable model for this purpose because chicken TRPV1 has been shown to be insensitive to capsaicin and thus the effects of capsaicin can be attributed to TRPV1-independent mechanisms. Administration of capsaicin (10 mg/kg, iv) to conscious unrestrained chicks at 5 days of age caused a transient decrease in body temperature. This effect of capsaicin was not observed in chicks that had been pretreated twice with capsaicin, indicating that the capsaicin-sensitive pathway can be desensitized. LPS (2 mg/kg, ip) induced fever that lasted for about 2.5 h, but fever was not induced in chicks that had been pretreated with capsaicin for 2 days. The preventive effect of capsaicin on LPS-induced fever was not blocked by capsazepine, an antagonist for TRPV1, but the antagonist per se blocked the febrile response to LPS. These findings suggest that a capsaicin-sensitive TRPV1-independent mechanism may be involved in LPS-induced fever.     

Cytoprotective and anticancer properties of coenzyme Q versus capsaicin

Coenzyme Q (CoQ) is an essential component of the mitochondrial electron transport chain and serves as an electron donor and acceptor in mitochondrial energy-linked respiration. CoQ1 was shown to prevent ROS formation and cell death in complex 1 inhibited cells. Low concentrations of capsaicin like CoQ1 inhibited ROS formation but CoQ1 was more effective at restoring the mitochondrial membrane potential collapse caused by complex 1 inhibitors such as rotenone. At low concentrations, capsaicin acts as a CoQ mimic by protecting against rotenone induced ROS formation and mitochondrial membrane potential collapse. Lipid peroxidation in isolated rat hepatocytes induced by cumene hydroperoxide and chloroacetaldehyde was also prevented. At higher concentrations, capsaicin and CoQ1 became cytotoxic. Hep G2 cells were more susceptible than hepatocytes. The cytotoxic mechanism for both capsaicin and CoQ1 was shown to involve a collapse of the mitochondrial membrane potential, however, only capsaicin caused ROS formation. The capsaicin side chain was required for capsaicin induced cytotoxicity. The anticancer properties of CoQ1 and capsaicin should prove useful for inducing tumor cell apoptosis.     

Repeated oral administration of capsaicin increases anxiety-like behaviours with prolonged stress-response in rats

This study was conducted to examine the psycho-emotional effects of repeated oral exposure to capsaicin, the principal active component of chili peppers. Each rat received 1 mL of 0.02% capsaicin into its oral cavity daily, and was subjected to behavioural tests following 10 daily administrations of capsaicin. Stereotypy counts and rostral grooming were significantly increased, and caudal grooming decreased, in capsaicin-treated rats during the ambulatory activity test. In elevated plus maze test, not only the time spent in open arms but also the percent arm entry into open arms was reduced in capsaicin-treated rats compared with control rats. In forced swim test, although swimming duration was decreased, struggling increased in the capsaicin group, immobility duration did not differ between the groups. Repeated oral capsaicin did not affect the basal levels of plasma corticosterone; however, the stress-induced elevation of plasma corticosterone was prolonged in capsaicin treated rats. Oral capsaicin exposure significantly increased c-Fos expression not only in the nucleus tractus of solitarius but also in the paraventricular nucleus. Results suggest that repeated oral exposure to capsaicin increases anxiety-like behaviours in rats, and dysfunction of the hypothalamic-pituitary-adrenal axis may play a role in its pathophysiology.     

Sympathetic activation by chemical stimulation of white adipose tissues in rats

Injection of leptin into white adipose tissue (WAT) increases sympathetic outflow. The present study was designed to determine the effects of capsaicin and other chemicals in WAT on the sympathetic outflow and blood pressure and the roles of WAT afferents and hypothalamic paraventricular nucleus (PVN) in the adipose afferent reflex (AAR). The AAR was induced by injection of capsaicin, bradykinin, adenosine, adenosine triphosphate (ATP), or leptin into inguinal WAT (iWAT) or retroperitoneal WAT (rWAT) in anesthetized rats. The iWAT injection of capsaicin increased the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) but not the heart rate. Bradykinin, adenosine, or leptin but not ATP in the iWAT caused similar effects to capsaicin on the RSNA and MAP. Intravenous, intramuscular, or intradermal injection of capsaicin had no significant effects on the RSNA and MAP. The effects of capsaicin in rWAT were similar to that in iWAT on the RSNA and MAP. Furthermore, injection of capsaicin into the iWAT increased the WAT afferent nerve activities, WAT efferent nerve activity, and brown adipose tissue efferent nerve activity. The iWAT denervation or chemical lesion of the PVN neurons with kainic acid abolished the AAR induced by the iWAT injection of capsaicin. These results indicate that the stimulation of iWAT afferents with capsaicin, bradykinin, adenosine, or leptin reflexly increases the RSNA and blood pressure. The iWAT afferents and the PVN are involved in the AAR induced by capsaicin in the iWAT.     

Small doses of capsaicin given intragastrically inhibit gastric basal acid secretion in healthy human subjects.

Although the direct inhibitory effect of small dose of capsaicin on gastric secretory responses was proved in animal observations, the role of capsaicin-sensitive afferent nerves (CSAN) and the effect of capsaicin applied in small and high doses on gastric secretion in human has not been clarified yet. In this study we investigated the influence of different small doses (100-800 microg) of capsaicin given intragastrically through an orogastric tube on gastric basal secretory responses in 10 healthy human subjects. Gastric basal secretory responses (volume, H+-concentration, H+-output) were measured from the suctions of gastric juice for a 1-h period. It has been found that: a) capsaicin dose-dependently inhibited the volume and H+-output of gastric juice; b) ID50 was found to be about 400 microg for capsaicin on gastric acid secretion; c) the time interval for capsaicin-induced gastric inhibition existed for about 1 h indifferently from the higher dose (800 microg) of capsaicin given after. It has been concluded that the capsaicin (given in small doses) inhibits the gastric basal acid output via stimulation of the inhibition of capsaicin sensitive afferent nerves.     

Effects of Capsaicin and Isoflavone on Blood Pressure and Serum Levels of Insulin-Like Growth Factor-I in Normotensive and Hypertensive Volunteers with Alopecia

Insulin-like growth factor-I (IGF-I) reduces arterial blood pressure. Since administration of capsaicin and isoflavone increases serum levels of IGF-I by sensory neuron stimulation in subjects with alopecia, it is possible that administration of capsaicin and isoflavone reduces arterial blood pressure in patients with hypertension. Systolic and diastolic blood pressure (BP) and serum levels of IGF-I were determined before and at 1, 3, and 5 months after administration of capsaicin and isoflavone in 42 volunteers with alopecia, 29 normotensive and 13 hypertensive volunteers. Neither systolic nor diastolic BP changed in the normotensive volunteers after combined administration of capsaicin and isoflavone. In contrast, systolic and diastolic BP was significantly reduced in hypertensive volunteers after administration of capsaicin and isoflavone. Serum levels of IGF-I significantly increased in both normotensive and hypertensive volunteers after administration of capsaicin and isoflavone. These observations suggest that administration of capsaicin and isoflavone might reduce BP in hypertensive, but not in normotensive subjects, probably by increasing serum levels of IGF-I.     

Capsaicin induces cofilin dephosphorylation in human intestinal cells: the triggering role of cofilin in tight-junction signaling

Previously, we demonstrated that capsaicin induces tight-junction (TJ) opening in human intestinal Caco-2 cells. In order to clarify the mechanism underlying the TJ opening action of capsaicin, we performed a proteomics study on capsaicin-treated Caco-2 cells. Phosphorylated cofilin was decreased significantly by capsaicin treatment. In addition, capsaicin induced Ca2+ influx in Caco-2 cells and there was a clear correlation between Ca2+) influx and cofilin dephosphorylation (activation). The Ca2+-chelating reagent EGTA blocked the cofilin dephosphorylation induced by both capsaicin and ionomycin, suggesting that the dephosphorylation was mediated by Ca2+ influx. Finally, transepithelial electrical resistance measurements showed that TJ opening accompanied cofilin dephosphorylation. Our data suggest that TJ opening is mediated by cofilin dephosphorylation, which is caused by capsaicin stimuli, including Ca2+ influx. This is the first report of capsaicin action via the dephosphorylation of cofilin in human intestinal cells.     

Oxidant-based anticancer activity of a novel synthetic analogue of capsaicin,capsaicin epoxide

Abstract

Objectives

Plant-derived natural substances, such as capsaicin, with potent antiproliferative activity against cancer cells in vitro are considered to be promising nutraceuticals in anticancer therapy. Nevertheless, the limited systemic bioavailability of phytochemicals may raise questions regarding the physiological relevance of their phytochemical effects in vivo. Thus, the search for novel phytochemical-based substances with more efficient anticancer action is needed.

Methods

In the present study, a capsaicin analogue, namely, capsaicin epoxide, was synthesized, and its cytotoxic potential against cancer cells was evaluated and compared to that of capsaicin through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and multi-caspase assays. The abilities of capsaicin and capsaicin epoxide to induce oxidative stress were estimated using redox-sensitive fluorogenic probes: 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA) and dihydroethidium.

Results

The structure and purity of the synthesized product were confirmed by nuclear magnetic resonance spectroscopy, electrospray ionization mass spectrometry, and gas chromatography. Normal human dermal fibroblasts were not susceptible to treatment with the agent, whereas a cancer cell type-specific response was observed. Human breast carcinoma cells were found to be the most sensitive to capsaicin epoxide treatment compared with capsaicin treatment, and the action of capsaicin epoxide was oxidant based.

Discussion

Our data indicate that the antiproliferative activity of capsaicin epoxide is potentiated in vitro, when used at much lower concentrations compared with capsaicin at similar concentrations. Thus, the findings of this study may have implications for phytochemical-based anticancer drug development.     

肾动脉内注射辣椒素兴奋肾神经传入纤维的自发活动

应用记录肾传入神经多单位和单位放电的方法,观察肾动脉内注射辣椒素对麻醉家兔肾神经传入纤维自发放电活动的影响。结果表明：(1)肾动脉内注射辣椒素20、40和60nmol／kg可呈剂量依赖性地兴奋肾传入纤维的活动,而动脉血压不变;(2)静脉内预先应用辣椒素受体阻断剂钌红(40mmol／kg),可完全阻断辣椒素对肾传人纤维的兴奋作用。(3)静脉内预先注射一氧化氮合酶抑制剂L-NAME(0．1mmol／kg),能延长并增强肾传入神经对辣椒素的反应。以上结果提示：肾动脉内应用辣椒素可兴奋肾传人纤维的自发放电活动。一氧化氮作为抑制因素参与辣椒素诱导的肾传入神经兴奋。     

Effects of oral capsaicin on gustatory, olfactory and irritant sensations and flavor identification in humans who regularly or rarely consume chili pepper

We explore interactions between the irritant effects of oralcapsaicin and gustatory and olfactory sensations, and the extentto which experience with chili pepper, and liking for its sensoryproperties are associated with changes in the perception oforal capsaicin. Oral capsaicin partially masks gustatory andolfactory sensations, but surprisingly, it does not interferewith flavor identification Regular users rate the intensityof orally-induced irritation from capsaicin as markedly lowerIn spite of this difference, the partial masking of the magnitudeof olfactory or gustatory sensations exerted by capsaicin isapproximately equal in the two groups. There are indicationsthat decrements in flavor identification under capsaicin aregreater in chili dislikers (non-eaters). The pattern of resultssuggests that the masking effect of capsaicin on taste and smellarises at the stage of processing before (or on a parallel pathto) the appreciation of the magnitude of the capsaicin-inducedburn sensation.     

Analysis of the mechanism of the tight-junctional permeability increase by capsaicin treatment on the intestinal Caco-2 cells

In a previous experiment (Isoda et al., 2001), we showed that the tight-junctional (TJ) permeability increase in Caco-2 cells during capsaicin exposure was through binding of the capsaicin molecule to a capsaicin receptor-like protein. In the present study, we examined how actin, which modulates TJ permeability, is influenced by capsaicin. We showed that after treatment of the Caco-2 cells with capsaicin, the volume of F-actin decreased. Moreover, we also examined protein kinase C (PKC) and heat shock protein 47 (HSP47), which act as probable second messengers in causing TJ permeability increase. We showed that after capsaicin treatment, HSP47 was activated. However, PKC activity was the same in both control and treatment setups. These results suggest that, while PKC is not involved, it is highly possible that HSP47plays a role in TJ permeability increase in intestinal Caco-2 cells exposed to capsaicin. This revised version was published online in August 2006 with corrections to the Cover Date.     

TRPV6 mediates capsaicin-induced apoptosis in gastric cancer cells--Mechanisms behind a possible new "hot" cancer treatment

Capsaicin is an organic compound in chili peppers which are consumed by over one quarter of the world's population daily. Studies have shown that capsaicin can induce apoptosis in some cancer cells by unknown mechanisms. In this study, both gastric cancer and normal epithelial cells were treated with capsaicin and examined for apoptosis by Annexin V binding. Our results showed that capsaicin induces apoptosis in both cells, although cancer cells are more susceptible. This susceptibility is dependent on the availability of TRPV6, a calcium-selective channel protein, as overexpression of TRPV6 in normal cells increased capsaicin-induced apoptosis and knockdown of TRPV6 in cancer cells suppressed this action. Our results further demonstrated that capsaicin increases mitochondrial permeability through activation of Bax and p53 in a JNK-dependent manner. Conclusions: (1) TRPV6, rather than TRPV1 (the well-known capsaicin receptor), mediates capsaicin-induced apoptosis in gastric cells; (2) abundance of TRPV6 in gastric cells determines their live or death under capsaicin treatment; and (3) capsaicin induces apoptosis by stabilization of p53 through JNK activation. Together, our data suggest that capsaicin may be a promising dietary candidate for cancer chemoprevention.