Black tea prevents cigarette smoke-induced apoptosis and lung damage

Abstract

Background: Cigarette smoking is a major cause of lung damage. One prominent deleterious effect of cigarette smoke is oxidative stress. Oxidative stress may lead to apoptosis and lung injury. Since black tea has antioxidant property, we examined the preventive effect of black tea on cigarette smoke-induced oxidative damage, apoptosis and lung injury in a guinea pig model.

Methods: Guinea pigs were subjected to cigarette smoke exposure from five cigarettes (two puffs/cigarette) per guinea pig/day for seven days and given water or black tea to drink. Sham control guinea pigs were exposed to air instead of cigarette smoke. Lung damage, as evidenced by inflammation and increased air space, was assessed by histology and morphometric analysis. Protein oxidation was measured through oxyblot analysis of dinitrophenylhydrazone derivatives of the protein carbonyls of the oxidized proteins. Apoptosis was evidenced by the fragmentation of DNA using TUNEL assay, activation of caspase 3, phosphorylation of p53 as well as over-expression of Bax by immunoblot analyses.

Results: Cigarette smoke exposure to a guinea pig model caused lung damage. It appeared that oxidative stress was the initial event, which was followed by inflammation, apoptosis and lung injury. All these pathophysiological events were prevented when the cigarette smoke-exposed guinea pigs were given black tea infusion as the drink instead of water.

Conclusion: Cigarette smoke exposure to a guinea pig model causes oxidative damage, inflammation, apoptosis and lung injury that are prevented by supplementation of black tea.

Figure 1

A, Oxyblot of lung proteins of guinea pigs exposed to air or cigarette smoke (CS) with or without giving black tea as the drink. The guinea pigs were exposed to air or cigarette smoke (as described under Materials and Methods) and were given water or black tea (BT) as the drink before being sacrificed after 7 days of CS/air exposure. Lane 1, air-exposed guinea pigs given BT as the drink; lane 2, CS-exposed guinea pigs given BT as the drink; lane 3, air-exposed guinea pigs given water as the drink; lane 4, CS-exposed guinea pigs given water as the drink. B, Densitometric measurement of the lanes 1, 2, 3, and 4, respectively of Figure 1 A using Quantity One- 4.4 (Bio-Rad) Software. * Bars over the respective columns represent means ± SD (n = 4).

Figure 2

Histopathology profiles of guinea pig lung tissue sections after exposure to air or cigarette smoke in the presence and absence of black tea. Marked enlargement of airspaces was found in lung sections of the guinea pigs in the CS group (see 'Materials and Methods'). The number of guinea pigs used in each group was 4. Eight images were analyzed in 4 lung sections (2 images/section/animal) from each group (magnification ×10). In sharp contrast to the CS-exposed groups (CS group), the enlargement of airspace was greatly reduced in the CS+BT group. The number of air spaces analyzed and the morphometric measurements with statistical difference between the groups are shown in Table 1. A, air-exposed guinea pigs given water as the drink (sham control); B, CS-exposed guinea pigs given water as the drink; C, air-exposed guinea pigs given BT as the drink; D, CS-exposed guinea pigs given BT as the drink.

Figure 3

A, Immunoblots of the DNP-derivatives of lung proteins of guinea pigs exposed to air or CS after day 1 and day 3. Twenty five μg protein isolated from air-exposed or CS-exposed guinea pigs were converted, without any further treatment, to the DNP-derivative followed by immunoblotting as mentioned in Materials and Methods. 1 and 3 mean exposed to air (sham control) or CS for 1 day and 3 days, respectively. B, C, Histopathology profiles of guinea pig lung tissue sections after exposure to cigarette smoke for 3 days. B shows infiltration of inflammatory cells in the septal regions. C shows accumulation of leukocytes within the alveolar cells that are in all probability macrophages (indicated by → ; magnification × 20)

Figure 4

Detection of DNA strand breaks in lung cells of guinea pigs exposed to air or CS in the presence or absence of BT by TUNEL assay. The guinea pigs were exposed to air or CS (as described under Materials and Methods) and sacrificed after 7 days of exposure. Lower Panel: the lung sections were stained with fluorescein labeled dUTP according to the protocols discussed under 'Materials and Methods'. A, guinea pigs exposed to air and given water as a drink; B, guinea pigs exposed to CS and given water as a drink; C, guinea pigs exposed to air and given BT as the drink; D, guinea pigs exposed to CS and given BT as the drink. Upper Panel: Lung sections corresponding to the upper panel were counterstained with DAPI to identify the cell nuclei.

Figure 5

Quantitative evaluation of TUNEL positive cells in lungs of guinea pigs exposed to air or CS in the presence or absence of BT. The percentage of TUNEL positive cells were measured from the results depicted in Figure 4. A,B,C,D, are same as in Figure 4. The number of animals, sections per animal and number of fields analyzed per section were 4, 4 and 2, respectively; the bars over the respective columns represent means ± SD (p < 0.05 between B and A, C or D).

Figure 6

Immunoblot of caspase 3 of the lung extracts of guinea pigs exposed to air or CS given water or BT as the drink. Upper Panel: lane 1, air-exposed guinea pigs given water; lane 2, CS-exposed guinea pigs given water; lane 3, air-exposed guinea pigs given BT; lane 4, CS-exposed guinea pigs given BT. Activation of caspase 3 is evidenced by the formation of cleaved caspase (17 kDa product). Lower panel: the membrane was re-probed with anti-mouse tubulin antibody to determine the level of tubulin as a loading control.

Figure 7

Immunoblot of phosphorylated p53 and p53 of lung extracts of guinea pigs exposed to air or CS given water or BT as the drink. Upper panel represents phosphorylated p53 (P-p53) and lower panel, p53. Lane 1, air-exposed guinea pigs given water; lane 2, CS-exposed guinea pigs given water; lane 3, air-exposed guinea pigs given BT; lane 4, CS-exposed guinea pigs given BT. Details of the experiment are given under 'Materials and Methods'.

Figure 8

Immunoblot of Bax and Bcl-2 of the lung extracts of guinea pigs exposed to air or CS given water or BT as the drink. Panel A and Panel B depict respective immunoblots of Bax and Bcl-2. Lane 1, air-exposed guinea pigs given water; lane 2, CS-exposed guinea pigs given water; lane 3, air-exposed guinea pigs given BT; lane 4, CS-exposed guinea pigs given BT. In each case the membrane was reprobed with anti-mouse tubulin antibody to determine the level of tubulin as a loading control. Panel C shows the Bax/Bcl-2 ratio observed in different groups.