Durk Pearson & Sandy Shaw’s®
Life Extension NewsTM
Volume 14 No. 5 • November 2011


Inhibition of Inducible Nitric Oxide Synthase Reverses Tobacco-Smoke Induced Emphysema in Mice

Chronic obstructive pulmonary disease (COPD) is already relatively common (with 80,000,000 suffering from the disease in 2005 and 3,000,000 dying from it at that time) and the World Health Organization warns that COPD may become the third leading cause of death by 2030 (which may be true, but we always take such long-term predictions with a large grain of salt, however, especially when they come from public or quasi-public—that is politically motivated—agencies with a vested interest in scaring people into putting more money into the agencies’ budgets.) But, getting back to the facts … COPD is becoming increasingly viewed not just as a respiratory disorder but as a systemic disease, involving skeletal muscle wasting, diaphragmatic dysfunction, and systemic inflammation.1 It is largely caused by inhalation of smoke from tobacco and cooking fires and of air pollutants from automotive, industrial, and power generation activities. The disease is progressive and treatments are relatively ineffective, with some patients requiring lung transplantation.

Recent studies in a mouse model of COPD, however, point the way to an effective therapy.1 Excitingly, the researchers publishing this paper found a way to protect mice from developing emphysema and pulmonary hypertension, but even to REVERSE established disease in the animals. Importantly, it was discovered that “… smoke exposure caused emphysema and pulmonary hypertension in wild-type mice and in mice lacking eNOS [the endothelial form of nitric oxide synthase], but not in mice lacking iNOS [inducible nitric oxide synthase].”1 In other words, iNOS was part of the process by which the smoke exposure caused emphysema and pulmonary hypertension. “Moreover, when the authors administered an iNOS inhibitor after 8 months of smoke exposure, the treatment reversed the lung damage within 3 months.”

A CURATIVE APPROACH

The substance used in the reported paper to inhibit iNOS was the iNOS inhibitor N6-(iminoethyl)-L-lysine (hereafter called L-NIL). The authors refer to their use of L-NIL to restore lung structure and function when wild-type mice were treated with L-NIL after FULL establishment of emphysema (i.e. initiation of L-NIL treatment after 8 months of chronic smoke exposure for an additional 3 month period) as a CURATIVE APPROACH, not the sort of description you see routinely used in highly respected journals such as Cell, in which this paper was published.

The authors suggest that ONOO– (peroxynitrite, formed by the chemical reaction of nitric oxide and superoxide radicals in mitochondria) “is a possible candidate for mediating the effects of iNOS upregulation on lung vasculature and parenchyma.”1 “The increased levels of nitrotyrosine, present as a possible consequence of ONOO– generation (as reported in ref 2), in WT mice following tobacco-smoke exposure were in accordance with our hypothesis that ONOO-upregulation is a key step in vascular remodeling and emphysema pathogenesis.”

More About ONOO– and Its Inhibition

ONOO– is chemically created by the reaction of nitric oxide with superoxide radicals, especially in the presence of a deficiency of arginine or a deficiency of the mitochondrial cofactor tetrahydrobiopterin. Under those conditions, you get was is called “uncoupling” in the mitochondria, where instead of generating nitric oxide from the interaction of arginine and nitric oxide synthase, the mitochondria generate superoxide radicals, which can lead to a plethora of adverse effects due to scavenging of nitric oxide (and the consequent reduced availability of nitric oxide) by superoxide and the powerful prooxidant effects of ONOO–.

A Natural Product That Downregulates iNOS in a Mouse Model of Asthma

As so frequently happens in the large scientific literature, more than one group is studying a particular phenomenon of interest at the same time, such as researchers seeking methods for controlling and producing the effects of iNOS. iNOS is an important part of the proinflammatory processes that, as part of the immune system, are key factors in preventing and fighting off infections. Hence, like so many other things, you do not want to inhibit iNOS too much, but just enough to prevent the adverse effects of excess inflammation. That balancing act is one reason that medicine is an art as well as a science. No two people will respond exactly the same to any pharmacological agent, such as an iNOS inhibitor, and thats why you need the help of an experienced doctor to help you use powerful medicinal agents.

As it happens, a paper was published in Planta Medica3 at almost the same time as the paper on the inhibition of iNOS as a potential curative approach to COPD (at least in mice). The new paper described the effects of grape seed proanthocyanidins (grapeseed proanthocyanidin extract, or GSPE) in a mouse model of asthma. As the authors report, nitric oxide (NO) plays an important role in the pathophysiology of asthma.3 “Increased NO level is found to be positively related to asthmatic airway inflammation and hyperresponsiveness. iNOS is also increased in asthmatic tissues, representing airway eosinophilic cells, T helper (Th) 2 cells, and mast cell infiltration.” Glucocorticoids (with their often powerful anti-inflammatory effects) are the first choice of asthma therapy, but “no significative curative effect is observed among those with severe asthma or steroidal resistance. In addition, years of steroid therapy cannot guarantee that asthmatic patients regain normal bronchial reactivity. It is therefore safe to say that an alternative anti-inflammatory treatment of asthma is badly needed.”

In the abstract of the new paper,3 the authors state that “[a]dministration of GSPE REMARKABLY suppressed airway resistance and reduced the total inflammatory cell and eosinophil counts in BALF.” See, for example, Fig. 3B. Also, Fig. 4B shows that infiltrating macrophages and neutrophils around the bronchi showed positive iNOS protein expression in the tissues of animals where inflammatory activation was induced by OVA (ovalbumin) as compared to animals not so sensitized. “GSPE treatment dramatically decreased them [the i-NOS stained cells in the OVA-induced inflammatory lung tissue].”

The authors make some comments on the safety of GSPE supplements. They note, for example, that a 3 month study of oral toxicity of 2.5% grape seed extract caused no adverse effects. Japanese researchers reported that no evidence showed acute oral toxicity in rats at dosages of 2 and 4 g/kg. “However, high-dose GSPE could cause apoptotic cell injury via effector caspase-3 activation and subsequent induction of reactive oxygen species generation.”2 (The general rule is that nothing is perfectly safe and that “the dose makes the poison.”) In their study,2 the authors found no significant cytotoxicity in GSPE-treated mice.

Other Natural Products Can Inhibit iNOS Under Certain Experimental Conditions

Quite a few studies (mostly in vitro)4 have identified natural products with anti-iNOS activity that include: naringenin (found in orange peel), kaempferol (found in broccoli and tea), quercetin (found in onion, broccoli, apples, and berries)—we include quercetin in our high potency multivitamin, multimineral, antioxidant formulation (128 mg in the daily recommended dosage of 12 capsules)—and silymarin. Our recent PubMed search of gamma tocopherol peroxynitrite also suggests that gamma tocopherol as protective against peroxynitrite (see, for example, refs 4b, 4c). Of course, to evaluate whether the iNOS inhibiting activity would be adequate to mitigate various disease conditions would require careful study of the literature for dosage used, type of cell cultures and/or animals involved, toxicity, and many other factors.

And, finally, as is mentioned in a recent paper,5 “[t]here are not however, many anti-inflammatory drugs that specifically target NOS or more specifically, iNOS, although there are various studies and trial that are investigating the effect of iNOS inhibition in various inflammatory conditions such as sepsis and other chronic illnesses including cancer.”

References

1. Seimetz et al. Inducible NOS inhibition reverses tobacco-smoke-induced emphysema and pulmonary hypertension in mice. Cell 147:293-305 (2011).
2. Szabo et al. Peroxynitrite: biochemistry, pathophysiology and development of therapeutics. Nat Rev Drug Discov 6:662-80 (2007).
3. Zhou et al. Grape seed proanthocyanidin extract attenuates airway inflammation and hyperresponsiveness in a murine model of asthma by downregulating inducible nitric oxide synthase. Planta Med 77:1575-81 (2011).
4. Pan et al. Modulation of inflammatory genes by natural dietary bioactive compounds. J Agric Food Chem 57:4467-77 (2009).
4b. McCarty. Gamma tocopherol may promote effective NO synthase function by protecting tetrahydrobiopterin from peroxynitrite. Med Hypotheses 69(6):1367-70 (2007).
4c. Ferroni et al. Phenolic antioxidants and the protection of low density lipoprotein from peroxynitrite-mediated oxidations at physiologic CO2. J Agric Food Chem 52(10):2866-74 (2004).
5. Liu et al. Targeting cyclooxygenase and nitric oxide pathway cross-talk: a new signal transduction pathway for developing more effective anti-inflammatory drugs. Curr Signal Transduct Ther 4:66-75 (2009).

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