Screening And Evaluation Of Two Natural Inhibitors Of TNF-α From Traditional Chinese Medicines In Vitro And In Vivo

Cytokines are small and non-structural cell-signaling protein molecules, which can be synthesized by nearly all nucleated cells. Moreover, cytokines play the key role in the contact not only among the immune system cells, but also between the immune system cells and other types of cells. By binding to the cell specific membrane receptors, cytokines change cells in their behavior and character. According to the different functions, cytokines have different classification. In infection and/or inflammatory reaction some cytokines clearly promote inflammation and are called pro-inflammatory cytokines, such as tumor necrosis factor-α(TNF-α) and IL-1, while other cytokines suppress the activity of pro-inflammatory cytokines and are called anti-inflammatory cytokines. This classification constructs the base of the inflammatory cytokine biology and their clinical medicine.TNF-αis mainly produced by activated monocytes/macrophages/T cells, which was first found to induce necrosis or apoptosis of tumor cells in the early study. Then TNF-αwas found to mediate specific inflammatory response and regulate immune function. Mainly through the TNF-αreceptor (TNF-R1) on the cell membrane TNF-αactivated the majority of biological activity, triggered a series of intramolecular events, eventually leaded to the activation of two key transcription factors, namely NF-κB and c-Jun. These two transcription factors can induce the expression of multiple genes to cause a number of different biological processes, including cell growth and death, tumor growth, immune, inflammatory and stress response. Inappropriate TNF-αproduction and durable activation of the signal will lead to a lasting wide range of human pathological processes, including the systemic inflammatory response syndrome (SIRS), inflammatory bowel disease (IBD), rheumatoid arthritis (RA), cerebral malaria, diabetes, cancer, bone osteoporosis, allograft rejection and multiple sclerosis.Since TNF-αis one of the initial and important mediators to stimulate the inflammatory process, the inhibition of its activity is the popular drug target in the world. Inhibitors of TNF-a had been used successfully in a variety of inflammatory diseases. For example, infliximab, etanercept and adalimumab has been approved by the U.S. FDA for the treatment of rheumatoid arthritis and Crohn's disease. In 2009 best-selling drugs in the world rankings, the three drugs above came into the top ten with separate sales of more than 50 billion U.S. dollars.Modern medical research and clinical practice has proved that many traditional Chinese medicines and their major active components showed the significant anti-inflammatory effect, the action may be related to their TNF-a inhibitory activity. Recent studies showed that ethyl acetate extract of angelica sinensis can improve the survival rate after LPS injection in mice with the reduction of TNF-a level in vivo, which may be related to the downregulation to the NF-κB activity. Another study reported that the main component of Angelica sinensis, ligustilide (accounting for more than 50% in the volatile oil), inhibited LPS-induced release of TNF-a in monocyte cell, with the inhibitory effect on mRNA transcription. Genetic analysis showed that ligustilide can inhibit TNF-a-mediated NF-κB activity.Systemic inflammatory response syndrome (SIRS) is the systemic reaction responding to the cytokines and inflammatory mediators, which is induced by infection or non-infection factors. Sepsis is a potentially deadly medical condition that is characterized by a whole-body inflammatory state and the presence of the known or suspected infection, severe cases can cause septic shock. Endotoxin shock is usually induced by Lipopolysaccharide (LPS) from the Gram-negative bacteria. TNF-a is an early released inflammatory mediator and play a central role in the endotoxin shock, which may promote other inflammatory mediators such as IFN-y, IL-6 and IL-12 rapidly, resulting in the so-called "cascade effect". The overproduction of TNF-a make the body in a high metabolic state and in a series of pathophysiological changes, such as fever, hypotension, lactic acidosis and irreversible shock. IL-1βis another important pro-inflammatory factor, and has a synergistic effect with TNF-a. So inhibition of TNF-a and IL-1βon the treatment of SIRS has a positive meaning.In view of inhibition of ligustilide on TNF-a release and the reported anti-inflammatory effects,, we hypothesized that it. may have a protective effect on SIRS, thus designed a series of experiments in vivo and in vitro to evaluate protective effect and action mechanism of ligustilide.Experiments confirmed that ligustilide mice inhibited the release of TNF-a on LPS-induced mouse leukaemic monocyte macrophage cell line (RAW264.7) with IC50 of 16.3μM, which indicated that ligustilide play anti-inflammatory activity possibly through inhibition of the release of TNF-a. To evaluate the protective effect of ligustilide we constructed two models in vivo including LPS-induced mouse shock and LPS-induced rabbit shock. Compared with the control group, ligustilide showed a protective effect on endotoxin shock of mice in the dose dependent manner, improving the survival rate of mice with endotoxic shock and prolonging of the average survival time.In this study, we also investigated the protective effects of ligustilide against lipopolysaccharide (LPS)-induced endotoxic shock in Japanese White Rabbits and attempted to elucidate the possible mechanism underlying these effects. Forty-two rabbits were randomized into 6 groups:normal group, LPS group, dexamethasone group (5 mg/kg), and 3 ligustilide groups (20,40, and 80 mg/kg). After the rabbits received LPS infusion (0.3 mg/kg), dexamethasone and ligustilide were intravenously injected at the abovementioned dosages. Heart rate (HR), mean arterial pressure (MAP), and rectal temperature (RT) were recorded throughout the experiment. Tumor necrosis factor-a (TNF-a), interleukin-1β(IL-1β), and nitric oxide (NO) levels were measured by radioimmunoassay every 30 minutes for the first hour, and every 60 minutes thereafter until the end of the experiment. The serum levels of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), y-glutamyl transpeptidase (GGT), creatinine kinase (CK), lactate dehydrogenase (LDH), total protein (TP), creatinine (Scr), blood urea nitrogen (BUN), total bilirubin (T. BIL), and counts of formed elements of blood were measured at 0,120, and 300 minutes after the administration of LPS. Hemorheology was assayed 300 minutes after the LPS injection. The vital organs were collected and weighed before histopathologic examination. A comparison between the LPS group and the ligustilide groups showed that ligustilide significantly inhibited the decline in MAP and RT and decreased the levels of TNF-a, IL-1β. and NO. but had no apparent effect on HR. Ligustilide also inhibited the increase in the levels of biochemical markers, such as ALT, AST, ALP, GGT, LDH, CK, BUN, and Scr, but showed no apparent effect on T. BIL and TP. Furthermore, ligustilide partly restored the function of injured vital organs, including the heart, liver, lungs, and kidneys. These results suggest that ligustilide protected the rabbits against LPS-induced endotoxic shock.Compared with the control group, ligustilide (high, medium and low-dose group) can inhibit not only xylene-induced ear edema swelling in mice significantly in a dose dependent manner, but also foot swelling induced by carrageenan, which indicated that ligustilide has a clear anti-inflammatory effect.The above experiments show ligustilide has a clear anti-inflammatory effect, especially show the protective effect on LPS-induced shock in rabbits and its mechanism may be the inhibition of TNF-α. All these suggest that ligustilide is worthy of further research and evaluation as a candidate drug for the treatment of systemic inflammatory response syndrome.Picrasma quassioides is another traditional anti-inflammatory medicine, used for clearing heat and dampness, toxic insecticide, antibacterial and anti-inflammatory, and also used for treatment of bacillary dysentery, gastroenteritis, biliary infection, acute purulent infection and other diseases. In contemporary time some prescriptions including extract of Picrasma quassioides are also used in clinic.The previous studies showed that the main chemical composition of Picrasma quassioides is a group of indole alkaloids.4-methoxy-5-hydroxycanthin-6-one is the main compound among these alkaloids so we further evaluate the anti-inflammatory effect of it on acute inflammatory model in vivo. Results showed that 4-methoxy-5-hydroxycanthin-6-one (12.5.25.50mg/kg) can significantly inhibit xylene-induced ear swelling in mice with the dose-dependent manner, and it also can inhibit carrageenan induced foot swelling in rats with the different dosage (6.25,12.5,25 mg/kg). These two experiments suggested that 4-methoxy-5-hydroxycanthin-6-one markedly inhibited the acute inflammation of both kinds of animals (mice, rats).Summarizing the above findings in vivo and in vitro and according to the report in the literature that Picrasma quassioides is often used to treat gastrointestinal tract disease in the modern clinical applications, we have established a dextran sulfate (DSS) induced model of ulcerative colitis to evaluate the protective effect of 4-methoxy-5-hydroxycanthin-6-one on ulcerative colitis. The results showed that oral administration of 4-methoxy-5-hydroxycanthin-6-one can significantly inhibit the decrease of body weight, reduce the severity of ulcers, alleviate the shrinkage of the colon, decrease myeloperoxidase (MPO) Activity in colon tissue, and inhibit the TNF-αlevel in serum.PDE 4. which can specifically hydrolyze cAMP. is the only intracellular enzymes for cAMP. Inhibition of PDE 4 characteristically rises the concentration of intracellular cAMP. The elevated intracellular cAMP concentration can reduced gene transcription in charge of encoding TNF-a in mononuclear cells. Our experiment showed that 4-methoxy-5-hydroxycanthin-6-one can inhibit PDE4 activity directly in vitro, which suggested that mechanism of the inhibition of TNF-a may be related to PDE4 inhibition activityIn conclusion, with the development of inflammatory diseases and inflammatory mechanisms in recent years, TNF-a, as one of the most important cytokines, is so prevalent in the drug research. The inhibitors of TNF-a have been used successfully for treatment of a variety of inflammatory diseases. Therefore, in our research we evaluated the effect of the main compounds from two traditional Chinese medicine angelica and Picrasma quassioides on the inhibitory activity of TNF-a in vivo and in vitro. Ligustilide showed the protective effect on SIRS and 4-methoxy-5-hydroxycanthin-6-one had the positive effect on UC, which suggested that the two compounds are worthy of further research and evaluation as drug candidates. Our research will also provide theoretical basis and data supports for preclinical studies of these two candidates.