Abdullatif Azab, Ahmad Nassar, Jacob Kaplanski, Reem Mahajneh, Galila Agam, Abed N.Azab,4?

1Triangle Research & Development Center, Kfar-Qari, 30026 Israel

2Formerly: Institute of applied Research, Box 437, Shefa-Amr, 20200 Israel

3Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

4Department of Nursing, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

1. Introduction

Notobasis syriaca(L.) Cass. (N. syriaca) is single species in the plant genusNotobasisof the Asteraceae family[1]. It is one of the most widespread plants in the eastern part of the Mediterranean,with spiny, beautiful flowers. In many regions it was used by humans and animals as nutritious food since antiquity. Archeological findings indicate the use ofN. syriacaas a food source by humans as early as 23 000 years ago[2]. Many of the medicinal and other activities of this plant are unknown, as compared to similarly common plants in Euroasia. For example, the chemical compositions and almost all medicinal properties ofMicromeria fruticosa[3],Origanum majorana[4],Salvia officinalis[5] andMalvasylvestris[6] are well established. Another notable difference can be easily observed betweenN. syriacaandSilybum marianum, a very closely related plant which mostly grows in the same habitats. The activities ofSilybum marianum-medicinal, anti-inflammatory and others - were extensively studied and its chemical composition is completely known[7,8]. The anti-inflammatory properties of many other plants were summarized in a recently published review[9].

N. syriacais a well-known plant to traditional medicine. In the Arab society, it has been used for treating headaches, plague,canker sores, vertigo and jaundice, and sometimes it is used to enhance lactation. It is also a component in some bitters formulas and an antioxidant. Fresh stem and leaf base are edible[10]. In many countriesN. syriacais used as a food[11]. For example, in Italy, the plant is used as a food with wide variety of forms: boiled, fried as vegetables with eggs and in omelets[12].

As mentioned above,N. syriacawas partially investigated by modern research. Antimicrobial activity of some extracts of the plant was reported by Ali-Shtayehet al.[13] but they did not indicate the extraction solvents and methods. On the contrary, total phenolic content and antioxidant activity of the plant were investigated by other groups that reported detailed extraction methods and solvents[14,15]. In the study by Alaliet al.[14] only aqueous extract was used, while in the study of El-Helaet al.[15], methanolic and aqueous extracts were reported. Expectedly, the aqueous extract was more active with higher phenolic content. Aqueous extract ofN. syriacawas found to have prominent antifungal activity[16], and natural products with antimicrobial activity were isolated fromPhomopsissp. fungi that lived on the plant[17].

The purpose of the present study was to examine the effects of an aqueous extract ofN. syriacaon bacterial endotoxin(lipopolysaccharide, LPS)-induced inflammation in rats.Systemic administration of LPS to animals results in an intense inflammatory response including prominent secretion of inflammatory constituents, abnormal changes in body temperature(BT), cardiovascular complications (such as reduced myocardial contractility and a decrease in blood pressure), organs failure (e.g.,acute kidney injury), among other pathological findings[18-24]. In mice and rats intraperitoneal (ip) administration of LPS induces a biphasic change in BT - an early decrease in BT (hypothermia)followed by elevation in BT (fever)[18,21,24-26]. The mechanism underlying the hypothermia caused by LPS is not fully understood.Many inflammatory components are thought to contribute to this intricate process; a central one of which is prostaglandin (PG)E2[18,21,24-26]. LPS-induced variations in BT are accompanied by increased levels of PGE2in the hypothalamus[18,21,24-26]. Of note,the thermoregulation zone is located in the hypothalamus in the anteroventral periventricular nucleus. Anti-inflammatory drugs that decrease hypothalamic production of PGE2attenuate LPS-induced hypothermia[27]. Moreover, it was found that psychotropic drugs(which are not classic anti-inflammatory drugs) significantly reduced LPS-induced hypothermia in rats, probably due to reduction of hypothalamic PGE2levels[24].

The present study tested the effects of pretreatment with an aqueous extract ofN. syriacaon plasma and brain levels of PGE2, interleukin(IL)-6 and tumor necrosis factor (TNF)-α in LPS-treated rats. In particular, we examined the levels of PGE2, IL-6 and TNF-α in three brain regions: hypothalamus (HT), hippocampus (HC) and frontal cortex (FC). PGE2affects various crucial physiological functions, including: endocrine system activity, cardiovascular and kidney function, excretion of neurotransmitters, immuneinflammatory activity, regulation of BT, among others[24,28-32]. IL-6 is a cytokine that exerts both anti-and pro-inflammatory activities;it regulates many physiological and pathological processes[33].TNF-α is a potent pro-inflammatory cytokine but it also confers some anti-inflammatory functions[34-36]. It is involved in numerous physiological as well as pathological conditions in humans and is thus a target for multiple therapeutic interventions[34-36]. In the brain,TNF-α affects the expression of many genes that are crucial for neurons function and survival[34].

2. Material and methods

2.1. Extraction of plant material

Aerial parts ofN. syriacawere air dried and ground to fine powder,and 500 g of the powder was soaked in distilled water for 24 h at 50 ℃. Then, it was filtered (Whatman 44) and the clear solution was concentrated by a rotary evaporator (50 ℃), yielding about 17 g of dry extract which was stored in deep freeze.

2.2. Animals

Male Sprague-Dawley rats were used all through the studies.Animals (weighing 225-250 g at experiments commencement)were housed three per cage and maintained under controlled environmental conditions (ambient temperature of (22±1) ℃, humidity 55%-60%, photoperiod cycle 12 h light: 12 h dark), fed regular Lab Chow and waterad libitum. Only animals with no signs of sickness were included in the studies. The procedures of the study were in accordance with the guidelines of the institutional Committee for the Use and Care of Laboratory Animals.

2.3. Measurement of body temperature

Rectal BT was measured with a plastic-coated thermometer(Anristu Meter Co., Japan). Rats were accustomed to BT measurement during three days before the beginning of the experiments.

2.4. Treatment with N. syriaca extract

After a week of acclimation to housing conditions and BT measurements, rats were orally treated with the dry extract ofN.syriacafor three successive days. On the evenings before each day of extract treatment, rats were deprived of food from 20:00 pm to 08:00 am. At 08:00, rats were allowed access to special containers containing either 2.5 g per rat of regular food, or, a total of 2.5 g per rat of regular food + dry extract ofN. syriaca(Extract). The extract was calculated per animal weight to give a dose of 500 mg/kg. Thus,each extract-treated rat received 500 mg/kg extract per day for 3 consecutive days. Rats were allowed access to the regular food or extract-containing containers for 2 h during which the animals consumed all amount of the food in the containers. Thereafter, all animals were allowed free access to regular food again. During the entire experimental procedure rats had free access to water.

2.5. Induction of inflammation by LPS

LPS fromEscherichia coliwas dissolved in sterile NaCl 0.9%solution. Rats were administered (ip) with LPS 1 mg/kg to induce an inflammatory response as described previously[24]. Control rats were injected ip with 0.2 mL NaCl 0.9%. LPS was administered at 2 h after food/extract consumption.

2.6. Blood collection and preparation of brain regions homogenates

BT was evaluated immediately before LPS injection (time zero)and at about 90 min thereafter. Then, rats were briefly anesthetized(with a mixture of 4% isoflurane in 100% oxygen) and immediately executed by decapitation. Blood was collected for plasma separation and brain regions (HT, HC and FC) were extracted and immediately frozen (-80 ℃). Thereafter, brain regions were weighed and homogenized in a homogenizing buffer (cold phosphate-buffered saline solution containing a cocktail of phosphatase and protease inhibitors purchased from Sigma) and centrifuged at 10 000g, 4 ℃for 10 min. Supernatants and pellets were separated, collected and immediately transferred to -80 ℃.

2.7. Determination of IL-6, PGE2 and TNF-α levels

Levels of IL-6, PGE2and TNF-α in plasma and brain samples(supernatants of homogenates) were measured using rat DuoSet ELISA kits (R&D Systems; Minneapolis, MN, USA) according to manufacturer's instruction. The detection limits of the assays were as follows: 39-2 500 pg/mL for PGE2; 62.5-4 000 pg/mL for TNF-α; and,125-8 000 pg/mL for IL-6. For determining plasma PGE2levels,samples were diluted 100 times to fit the standard curve of the assay.In all ELISA experiments, when the level of the tested inflammatory constituent was under the lower detection limit of the assay, results were marked as "undetectable" and calculated as zero.

2.8. Statistical analysis and presentation of the data

Statistical evaluations were carried out using two-tailed Student’st-test. Results are presented as mean±SEM for the sample size as indicated in each table. Values ofP＜0.05 were considered statistically significant. The data express the results of one experiment out of three independent experiments with similar results. Results of plasma IL-6, TNF-α and PGE2levels are presented as pg/mL according to ELISA results. Results of IL-6, TNF-α and PGE2levels (homogenates of brain samples) were calculated as follows:ELISA result (pg/mL) divided by weight of each brain sample; data are presented as pg/(mL·mg wet weight of brain sample).

3. Results

3.1. Effects of N. syriaca on LPS-induced hypothermia in rats

Administration of LPS led to a significant hypothermia in rats. The BT of the control rats, extract-treated rats, LPS-treated rats and LPS +Extract-treated rats was (0.08±0.13), (0.24±0.14), (-1.16±0.26),and (-0.61±0.12) ℃, respectively. Pretreatment withN. syriacaextract alone slightly but significantly increased BT of treated animals (P＜0.05). Moreover, pretreatment withN. syriacaextract significantly attenuated LPS-induced hypothermia in rats (P＜0.05).

3.2. Effects of N. syriaca on IL-6, TNF-α and PGE2 levels in the plasma of LPS-treated rats

As seen in Table 1, IL-6 was undetectable in plasma of control and extract (alone)-treated animals. On the other hand, LPS led to a significant elevation in plasma IL-6 levels (P＜0.05). Pretreatment withN. syriacaextract did not significantly decrease LPS-induced elevation in IL-6 levels. Similarly TNF-α was undetectable in plasma of control and extract-treated animals (Table 1) and pretreatment withN. syriacadid not significantly alter LPS-induced elevation in plasma TNF-α levels (Table 1). As for PGE2, its levels did not differ significantly between control animals and those treated withN. syriacaextract alone (Table 1). LPS significantly increased its plasma levels (P＜0.05) and pretreatment withN. syriacaled to a nonsignificant decrease in PGE2levels in LPS-treated rats (Table 1).

Table 1Effects of N. syriaca on plasma IL-6, TNF-α and PGE2 levels rats (pg/mL).

3.3. Effects of N. syriaca on IL-6, TNF-α and PGE2 levels in the hypothalamus of LPS-treated rats

Administration of LPS and pretreatment withN. syriacaalone did not significantly alter IL-6 and TNF-α levels in the HT (Table 2). On the other hand, pretreatment withN. syriacasignificantly decreased IL-6 and TNF-α levels in HT of LPS-treated rats(P＜0.05) (Table 2). Consistent with previous studies, administration of LPS led to a significant increase in HT PGE2levels (P＜0.05)(Table 2). Pretreatment withN. syriacaalone did not alter PGE2levels in this brain region, whereas pretreatment withN. syriacasignificantly decreased HT PGE2levels in LPS-treated rats (P＜0.05)(Table 2).

3.4. Effects of N. syriaca on IL-6, TNF-α and PGE2 levels in the hippocampus of LPS-treated rats

Administration of LPS did not significantly alter IL-6 and PGE2levels in the HC (Table 2). In contrast, LPS significantly decreased HC TNF-α levels (P＜0.05) (Table 2). Pretreatment withN. syriacasignificantly reduced IL-6 and TNF-α levels in the HC of control as well as LPS-treated rats (P＜0.05) (Table 2). Similarly, pretreatment withN. syriacasignificantly decreased HC PGE2levels in LPS-treated rats (P＜0.05) (Table 2).

3.5. Effects of N. syriaca on IL-6, TNF-α and PGE2 levels in the frontal cortex of LPS-treated rats

Neither administration of LPS nor pretreatment withN. syriacaalone did not affect IL-6, TNF-α and PGE2levels in the FC (Table 2). Unexpectedly, pretreatment withN. syriacasignificantly elevated IL-6 and TNF-α levels in the FC of LPS-treated rats (P＜0.05)(Table 2) and pretreatment withN. syriacaled to a non-significant increase in PGE2levels in the FC of LPS-treated rats (Table 2).

Table 2Effect of N. syriaca on hypothalamus IL-6, TNF-α and PGE2 levels in rats [pg/(mL·mg wet weight of brain sample)].

4. Discussion

To the best of our knowledge, the present study is the first to demonstrate thatN. syriacaexerts protective effects against LPS-induced inflammation. We found that oral pretreatment with a dry extract ofN. syriacafor 3 d resulted in significant anti-inflammatory effects in LPS-treated rats (P＜0.05), including: i) Attenuation of LPS-induced hypothermia; ii) reduction of hypothalamic and hippocampal PGE2levels in LPS-treated rats; and, iii) decrease of IL-6 and TNF-α levels in HT and HC of LPS-treated rats.Nevertheless, pretreatment withN. syriacaalso led to some proinflammatory effects such as elevation of IL-6 and TNF-α in FC of LPS-treated rats.

In general, the results of the present study suggest thatN. syriacaconfers anti-inflammatory effects, and that chronic consumption of the plant may be associated with anti-inflammatory and probably other protective properties. It is worth noting that this study utilized a sub-chronic treatment protocol - animals were treated with the extract once daily for 3 consecutive days. It is possible that a longer treatment-duration would have resulted in more profound antiinflammatory effects. However, this assumption remains to be confirmed or alternatively refuted. Further study is also necessary to determine the dose-dependent effect ofN. syriacaon the inflammatory parameters that were examined in this study. Moreover,the choice of oral (in the food) administration of the extract was done purposely to resemble a naturally occurring consumption of the plant materials. We decided against administering the extract through oral lavage or parenteral administration because these routes would not have mimicked a natural consumption of the plant.

The protective effect ofN. syriacaagainst LPS-induced hypothermia resembles that of classic anti-inflammatory drugs such non-steroidal anti-inflammatory drugs[27]. These drugs attenuate LPS-induced hypothermia primarily due to inhibition of PGE2production in the HT[27]. Consistently, psychotropic drugs, that are not classic antiinflammatory compounds, were also capable of reducing LPS-induced hypothermia in rats, probably due to decreasing HT PGE2levels[24]. Therefore, we speculate thatN. syriacaalso attenuated LPS-induced hypothermia due to inhibition of PGE2production in the HT. Importantly, the protective effect ofN. syriacaagainst LPS-induced hypothermia was observed in all three experiments that we performed. On the other hand, currently we do not understand the reason for the slight elevation in BT which was seen in rats that were treated only withN. syriaca. Although these animals did not show any visible pathological characteristic (anhedonia - as could be featured by reduction in social activity or decrease in food and water consumption, weight loss or diarrhea), this result was seen in two of the three experiments that we performed.

The precise mechanism underlying the protective effects ofN. syriacaagainst LPS-induced inflammation is currently not known. We speculated thatN. syriacamay alter the activity of the transcription factor nuclear factor kappa B (NF-κB), as was reported for other plant products with anti-inflammatory activities[9]. NF-κ B regulates the activity of numerous cellular pathways such as those involved immune and inflammatory responses, cancer pathophysiology,among others[37-40]. The mammalian NF-κB pathway involves the activity of a number of proteins such as p50, p65, and RelB, which contribute to various physiological and pathological processes[37-39].Different stimuli activate the NF-κB pathway including infectious microorganisms, LPS, cytokines, tissue damage, neurodegeneration and ischemia[37-39]. These stimuli activate the pathway by inducing phosphorylation and subsequent degradation of NF-κB inhibitor proteins (e.g., IκBα and IκBβ) which inhibit the translocation of NF-κB proteins to the nucleus. After dissociation of NF-κB proteins from IκB, they undergo phosphorylation by other kinases and then translocate to the nucleus and facilitate transcription of target genes. Therefore, it is recognized that elevated levels of phosphorylated NF-κB proteins (such as p65) in the nucleus represent increased activity of the pathway[24,37,39]. In this regard,we conducted a preliminary experiment in which we examined the effect ofN. syriacaon levels of nuclear phosphorylated-p65, as we described previously[24]. We found that pretreatment withN. syriacafor 3 d did not at all affect nuclear phosphorylated-p65 levels in HT and HC of LPS-treated rats (data not shown). These preliminary results suggest that the anti-inflammatory effects ofN. syriacaprobably derive from a non-NF-κB-associated mechanism.

This study demonstrated that an aqueous extract ofN. syriacaexhibited anti-inflammatory effects in LPS-treated rats. Further study is necessary to elucidate the mechanism underlying the antiinflammatory effects ofN. syriacaand the appropriate treatment regimen, including determination of the most efficacious dose and treatment duration. Moreover, further research is warranted to determine the chemical composition of the extract and reveal its most biologically active chemical constituents.

Conflict of interest statement

We declare that we have no conflict of interest.

[1] United States Department of Agriculture. U.S National Plant Germplasm System. [Online]. Available online: https://npgsweb.ars-grin.gov/gringlobal/taxonomydetail.aspx?id=447370. Accessed on March 1, 2017.

[2] Snir A, Nadel D, Groman-Yaroslavski I, Melamed Y, Sternberg M, Bar-Yosef O, et al. The origin of cultivation and proto-weeds long before Neolithic farming.PLoS One2015; 10: e0131422.

[3] Azab A. Micromeria: Chemistry and medicinal activies.Eur Chem Bull2016; 5: 300-307.

[4] Wahby MM, Yacout G, Kandeel K, Awad D. LPS-induced oxidative inflammation and hyperlipidemia in male rats: The protective role ofOriganum majoranaextract,BENI-SEUF Univ J Appl Sci2015; 4: 291-298.

[5] Khedher MRB, Khedher SB, Chaieb I, Tounsi S, Hammami M. Chemical composition and biological activities ofSalvia officinalisessential oil from Tunisia.EXCLI J2017; 16: 160-173.

[6] Benso B, Rosalen PL, Alencar SM, Murata RM.Malva sylvestrisinhibits inflammatory response in oral human cells. Anin vitroinfection model.PLoS One2015; 10: e0140331.

[7] Hermenean A, Stan M, Ardelean A, Pilat L, Mihali CV, Popescu C, et al. Antioxidant and hepatoprotective activity of milk thistle (Silybum marianumL. Gaertn.) seed oil.Open Life Sci2015; 10: 147-158.

[8] Qin NB, Jia CC, Xu J, Li DH, Xu FX, Bai J, et al. New amides from seeds ofSilybum marianumwith potential antioxidant and antidiabetic activities.Fitoterapia2017; 119: 83-89.

[9] Azab A, Nassar A, Azab AN. Anti-inflammatory activity of natural products.Molecules2016; 21: 1321.

[10] Qasem JR. Prospects of wild medicinal and industrial plants of saline habitats in the Jordan valley.Pak J Bot2015; 47: 551-570.

[11] Della A, Paraskeva-Hadjichambi D, Hadjichambis AC. An ethnobotanical survey of wild edible plants of Paphos and Larnaca countryside of Cyprus.J Ethnobiol Ethnomed2006; 2: 34.

[12] Licata M, Tuttolomondo T, Leto C, Virga G, Bonsangue G, Cammalleri I, et al. A survey of wild plant species for food use in Sicily (Italy) - results of a 3-year study in four Regional Parks.J Ethnobiol Ethnomed2016; 12: 12.

[13] Ali-Shtayeh MS, Al-Assali AA, Jamous RM. Antimicrobial activity of Palestinian medicinal plants against acne-inducing bacteria.Afr J Microbiol Res2013; 7: 2560-2573.

[14] Alali FQ, Tawaha K, El-Elimat T, Syouf M, El-Fayad M, Abulaila K, et al.Antioxidant activity and total phenolic content of aqueous and methanolic extracts of Jordanian plants: An ICBG project.Nat Prod Res2007; 21: 1121-1131.

[15] El-Hela AA, Abdel-Hady NM, Dawoud G, Hamed A, Morsy T. Phenolic content, antioxidant potential andAeded aegyptiiecological friend larvicidal activity of some selected Egyptian plants.J Egypt Soc Parasitol2013; 43: 215-234.

[16] Qasem JR, Abu-Blan HA. Fungicidal activity of some common weed extracts against different plant pathogenic fungi.J Phytopathology1996;144: 157-161.

[17] Hussain H, Tchimene MK, Ahmed I, Meier K, Steinert M, Draeger S,et al. Antimicrobial chemical constituents from the endophytic fungusPhomopsissp. fromNotobasis syriaca.Nat Prod Commun2011; 6: 1905-1906.

[18] Kaplanski J, Nassar A, Sharon-Granit Y, Jabareen A, Kobal SL, Azab AN. Lithium attenuates lipopolysaccharide-induced hypothermia in rats.Eur Rev Med Pharmacol Sci2014; 18: 1829-1837.

[19] Petroni RC, Biselli PJ, Lima TM, Velasco IT, Soriano FG. Impact of time on fluid resuscitation with hypertonic saline (NaCl 7.5%) in rats with LPS-induced acute lung injury.Shock2015; 44: 609-615.

[20] Vaez H, Rameshrad M, Najafi M, Barar J, Barzegari A, Garjani A.Cardioprotective effect of metformin in lipopolysaccharide-induced sepsis via suppression of toll-like receptor 4 (TLR4) in heart.Eur J Pharmacol2016; 772: 115-123.

[21] Matsuwaki T, Shionoya K, Ihnatko R, Eskilsson A, Kakuta S, Dufour S,et al. Involvement of interleukin-1 type 1 receptors in lipopolysaccharideinduced sickness responses.Brain Behav Immun2017; pii: S0889-1591(17)30202-7.

[22] Mirzoyan K, Denis C, Casemayou A, Gilet M, Marsal D, Goudounéche D, et al. Lysophosphatidic acid protects against endotoxin-induced acute kidney injury.Inflammation2017; 40: 1707-1716.

[23] Yaroustovsky M, Abramyan M, Komardina E, Nazarova H, Popov D, Plyushch M, et al. Selective LPS adsorption using polymyxin B-immobilized fiber cartridges in sepsis patients following cardiac surgery.Shock2017; doi: 10.1097/SHK.0000000000001016.

[24] Nassar A, Sharon-Granit Y, Azab AN. Psychotropic drugs attenuate lipopolysaccharide-induced hypothermia by altering hypothalamic levels of inflammatory mediators in rats.Neurosci Letters2016; 626: 59-67.

[25] Emílio-Silva MT, Mota CMD, Hiruma-Lima CA, Antunes-Rodrigues J, Cárnio EC, Branco LGS. Antipyretic effects of citral and possible mechanisms of action.Inflammation2017; doi: 10.1007/s10753-017-0615-4.

[26] Mota CMD, Rodrigues-Santos C, Fernández RAR, Carolino ROG,Antunes-Rodrigues J, Anselmo-Franci JA, et al. Central serotonin attenuates LPS-induced systemic inflammation.Brain Behav Immun2017; pii: S0889-1591(17)30217-9.

[27] Azab AN, Kobal S, Rubin M, Kaplanski J. Inhibition of prostaglandins does not reduce the cardiovascular changes during endotoxemia in rats.Prostaglandins Leukot Essen Fatty Acids2006; 74: 135-142.

[28] N?rregaard R, Kwon TH, Fr?ki?r J. Physiology and pathophysiology of cyclooxygenase-2 and prostaglandin E2in the kidney.Kidney Res Clin Pract2015; 34: 194-200.

[29] Sugimoto Y, Inazumi T, Tsuchiya S. Roles of prostaglandin receptors in female reproduction.J Biochem2015; 157: 73-80.

[30] Yang G, Chen L. An update of microsomal prostaglandin E synthase-1 and PGE2receptors in cardiovascular health and diseases.Oxid Med Cell Longev2016; 2016: 5249086.

[31] Sander WJ, O'Neill HG, Pohl CH. Prostaglandin E2as a modulator of viral infections.Front Physiol2017; 8: 89.

[32] Singh AK, Zajdel J, Mirrasekhian E, Almoosawi N, Frisch I, Klawonn AM, et al. Prostaglandin-mediated inhibition of serotonin signaling controls the affective component of inflammatory pain.J Clin Invest2017; 127: 1370-1374.

[33] Liu X, Jones GW, Choy EH, Jones SA. The biology behind interleukin-6 targeted interventions.Curr Opin Rheumatol2016; 28: 152-160.

[34] Probert L. TNF and its receptors in the CNS: The essential, the desirable and the deleterious effects.Neuroscience2015; 302: 2-22.

[35] Abbott R, Whear R, Nikolaou V, Bethel A, Coon JT, Stein K, et al.Tumour necrosis factor-α inhibitor therapy in chronic physical illness:A systematic review and meta-analysis of the effect on depression and anxiety.J Psychosom Res2015; 79: 175-184.

[36] Levy RA, Guzman R, Casta?eda-Hernández G, Martinez-Vazquez M,Damian G, Cara C. Biology of anti-TNF agents in immune-mediated inflammatory diseases: Therapeutic implications.Immunotherapy2016;8: 1427-1436.

[37] Oeckinghaus A, Hayden MS, Ghosh S. Crosstalk in NF-κB signaling pathways.Nat Immunol2011; 12: 695-708.

[38] Pozniak PD, White MK, Khalili K. TNF-α/NF-κB signaling in the CNS:Possible connection to EPHB2.J Neuroimmune Pharmacol2014; 9: 133-141.

[39] Zhu J, Cynader MS, Jia W. TDP-43 inhibits NF-κB activity by blocking p65 nuclear translocation.PLoS One2015; 10: e0142296.

[40] Kopitar-Jerala N. Innate immune response in brain, NF-kappa B signaling and cystatins.Front Mol Neurosci2015; 8: 73.