INTRODUCTİON

Isoniazid and rifampicin are anti-mycobacterial drugs that are part of the World Health Organisation’s recommended combination treatment regimen for tuberculosis¹. Treatment for tuberculosis consists of a quartet of anti-mycobacterial medications, including isoniazid, rifampicin, ethambutol, and pyrazinamide. Furthermore, the isoniazid and rifampicin combination (IRC) includes the supportive phase of tuberculosis treatment ^1,2. Although effective in treating tuberculosis, these drugs may cause serious side effects. During treatment, there may be toxic effects on the dermatology, gastrointestinal system, hypersensitivity, neurology, hematology, and renal system ³. Antituberculosis drugs can cause liver toxicity, which can result in the discontinuation of the drug as well as morbidity and mortality ⁴. A primary antituberculosis drug, isoniazid, has been reported to cause liver injury through oxidative stress ⁵. The role of lipid peroxidation (LPO) in the pathogenesis of isoniazid-associated oxidative liver injury has been documented ⁶. Severe oxidative liver damage is believed to be initiated by reactive oxygen species (ROS) ⁷. According to the literature, isoniazid has hepatotoxic effects due to the formation of the acetylhydrazine metabolite due to its acetylation ¹. Although rifampicin alone has a low hepatotoxicity potential ⁶, it exhibits additive and synergistic hepatotoxicity when used with isoniazid in treating tuberculosis ⁴. This has been attributed to the stimulation of hydrolases by rifampicin and the formation of hepatotoxic reactive metabolites from isoniazid ⁸.

The protective effect of thiamine pyrophosphate (TPP), thiamine’s active metabolite, was evaluated in IRC-induced hepatotoxicity in rats ⁹. Evidence in the literature shows that TPP protects various organs from oxidative and proinflammatory cytokine damage ^10-12. Cinnamon, which we also investigate in our study against the possible toxicity of IRC on the liver, is the bark of some Cinnamomum (Lauraceae) species ¹³. Cinnamon has been shown to have anti-inflammatory, antioxidant, and many other beneficial biological properties in the literature ¹⁴. It has been reported that cinnamon protects the heart, liver, kidney, blood, brain, and spleen from the toxicity of chemicals through its antioxidant, radical-scavenging, and LPO-suppressing properties¹⁵. The anti-inflammatory effect of cinnamon extract (CE) has been associated with inhibition of proinflammatory cytokines ¹⁶. This information suggests that TPP and CE may be helpful in treating IRC-induced liver injury. This study was designed to investigate the protective effects of TPP and CE against IRC-induced liver injury in rats.

MATERİALS AND METHODS

RESULTS

Analysis of MDA, tGSH, SOD and CAT levels in liver tissue

It can be seen from Fig. 1A that MDA levels in liver tissue increased in the IRG group (5.48±0.20) compared to healthy rats (2.39±0.18) (p<0.001). The increase in MDA was observed to be suppressed in the TIRG (2.67±0.24) and CIRG (4.06±0.15) (p<0.001). Compared with CE, TPP suppressed the increase in MDA more effectively (p<0.001). The MDA levels in TIRG and HG were not significantly different (p=0.100).

Fig. 1(A-D) Results of analyses of MDA (A), tGSH (B), SOD (C) and CAT (D) data measured from liver tissues. *; p<0.001 vs IRG, # p>0.05 vs HG. MDA: malondialdehyde, tGSH: total glutathione, SOD: superoxide dismutase, CAT: catalase, HG: healthy group, IRG: isoniazid+rifampicin applied group, TIRG: thiamine pyrophosphate+izoniazid+rifampisin applied group, CIRG: Cinnamon extract+izoniazid+rifampisin applied group. 

In IRG, a decrease in tGSH levels and SOD and CAT activities (3.42±0.24, 5.33±0.15, 3.78±0.12, respectively) were detected with the increase in MDA compared to HG (6.34±0.18, 9.19±0.12, 7.51±0.21, respectively) (p<0.001). TPP (5.80±0.10, 8.76±0.13, 7.09±0.26, respectively) and CE (4.48±0.09, 6.66±0.16, 5.39±0.37, respectively) inhibited the decrease in tGSH levels and SOD and CAT activities (p<0.001). TPP treatment prevented the decrease in antioxidants more effectively than CE (p<0.001) (Fig. 1B-D).

Analysis of NF-κB, TNF-α, IL-1β and IL-6 levels in liver tissue

As presented in Fig. 2A-D, the levels of NF-κB, TNF-α, IL -1β, and IL -6 in the liver tissues of rats in the IRG group (4.86±0.09, 4.68±0.09, 6.29±0.15, 5.70±0.12, respectively) were higher than those in the HG group (2.38±0.11, 2.13±0.07, 3.37±0.19, 2.49±0.24, respectively) (p<0.001). Compared to the IRG group, both TPP (2.57±0.12, 2.37±0.11, 3.68±0.26, 2.77±0.11, respectively) and CE (3.25±0.15, 3.31±0.14, 5.00±0.48, 4.02±0.22, respectively) inhibited these increases in NF-κB, TNF-α, IL -1β and IL -6 (p<0.05). However, this inhibition was more significant in the TPP than in the CE group (p<0.05). There were similarities between TIRG and HG in terms of NF-κB (p=0.315), IL -1β (p=0.157) and IL -6 (p=0.064) levels.

Fig. 2 (A-D) Results of analyses of NF-κB (A), IL -1β (B), TNF-α (C), and IL -6 (D) data measured from liver tissues. *; p<0.05 vs IRG, #, p>0.05 vs HG. NF-κB: nuclear factor kappa-B, IL -1β: interleukin 1-beta, TNF-α: tumor necrosis factor-alpha, IL -6: interleukin-6, HG: healthy group, IRG: isoniazid+rifampicin group, TIRG: thiamine pyrophosphate+izoniazid+rifampisin group, CIRG: Cinnamon extract+izoniazid+rifampisin group. 

Analysis of ALT and AST activities in serum samples

As presented in Fig. 3A-B, ALT and AST activities measured from the serum of rats in the IRG (86.67±4.55, 220.00±7.72, respectively) were found to be increased according to HG (29.17±4.02, 39.17±4.92, respectively) (p<0.001). The increase in ALT and AST activities in the IRG appeared to be suppressed in TIRG (32.83±3.55, 45.83±4.62, respectively) and CIRG (56.50±2.43, 92.50±4.51, respectively) (p<0.001). TPP inhibited this increase in ALT and AST activities more effectively than CE (p<0.001). Serum ALT (p=0.165) and AST (p=0.200) activities of TIRG and HG were similar.

Fig. 3 (A-B) Results of analyses of ALT (A) and AST (B) data measured from serum. *; p<0.001 vs IRG, #, p>0.05 vs HG. ALT: alanine aminotransferase, AST: aspartate aminotransferase HG: healthy group, IRG: isoniazid+rifampicin group, TIRG: thiamine pyrophosphate+izoniazid+rifampisin group, CIRG: cinnamon extract+izoniazid+rifampisin group. 

Histopathologic findings

There were normal central arteries in the liver tissue sections, radially arranged hepatic cords in the lobules, and normal tissue structure in the HG group, as presented in Fig. 4A and Table 1.

Fig. 4 (A-D) A. Liver tissue belonging to the HG. ; hepatocyte, ; Kupffer cell, ; normal appearance of blood vessels (H&E x200). B. Liver tissue belonging to the IRG. ; degenerated hepatocyte, ; irregularity in hepatic cords, ; increased Kupffer cells, ►; polymorphonuclear cell infiltration, ; moderately congested blood vessel (H&E x 200). C. Liver tissue belonging to the TIRG. ; hepatocyte with normal morphology, : Kupffer cell, ; slightly congested blood vessel (H&E x 200). D. Liver tissue belonging to the CIRG. ; moderately degenerated hepatocytes, ; increased Kupffer cells, ; irregularity in the hepatic cords, ►; weakly polymorphonuclear cell infiltration, ; moderately congested blood vessels (H&E x200). HG: healthy group, IRG: isoniazid+rifampicin group, TIRG: thiamine pyrophosphate+izoniazid+rifampisin group, CIRG: Cinnamon extract+izoniazid+rifampisin group. 

Table 1 Analysis results of histopathological grading data of liver tissues from rats. 

Parameters	Groups (n=6/each groups)				H / p values
	HG (n=6)	IRG (n=6)	TIRG (n=6)	CIRG (n=6)
	Median (quartile 1-quartile 3)
Degeneration	0(0-0)*	3(2-3)	0(0-0)*,#	2(2-2)£	129.013<0.001
Kupffer cell activation	0(0-0)*	3(2-3)	0(0-0)*,#	2(2-2)£	129.151/<0.001
Congestion	0(0-0)*	2(2-3)	0.5(0-1)*,#	2(2-2)£	122.132/<0.001
PMNL infiltration	0(0-0)*	3(3-3)	1(1-1)*	1(0.25-2)*	114.468/<0.001

Histopathological grading (0-3): 0: absent, 1: mild damage, 2: moderate damage, 3: severe damage. * p<0.001 vs. IRG, #; p>0.05 vs. HG, £; p>0.05 vs. IRG, HG: healthy group, IRG: isoniazid+rifampicin group, TIRG: thiamine pyrophosphate + izoniazid + rifampisin group, CIRG: Cinnamon extract + izoniazid + rifampisin group. Statistical analysis was done using the Kruskal-Wallis test. Then, Dunn’s test was applied. Data are presented as median (quartile 1-quartile 3). p<0.05 was considered significant.

Upon examination of the liver tissue of the IRG group, it was observed that the hepatocytes around the central artery had lost their typical morphology, were degenerating, their nuclei bulged and appeared pyknotic on occasion. A noticeable phenomenon was the loss of radial arrangement of hepatic cords, which resulted in irregular and spiralized cords. There was a marked and moderate degree of congestion in both the central artery and surrounding blood vessels. A significant increase was observed in the number of Kupffer cells. Many PMNLs were detected infiltrating the pericapillary area (Fig. 4B and Table 1).

Notably, in the TIRG group treated with TPP, the arrangement of hepatic cords and hepatocytes was generally normal, and the Kupffer cell population was relatively similar to the control group. PMNL infiltration was rare throughout the tissue (Fig. 4C and Table 1).

In the CE-treated CIRG group, it was remarkable that hepatic cords were irregular, and hepatocyte degeneration was at a moderate level. In general, it was observed that the congestion in the blood vessels was at a moderate level, and the Kupffer cell population was increased. Mild PMNL infiltration was also determined in the pericapillary area (Fig. 4D and Table 1).

DİSCUSSİON

The primary treatment protocol for tuberculosis consists of isoniazid, rifampicin, pyrazinamide, and ethambutol for six months ¹⁸. Increased dosages of these drugs have been tried to overcome resistance, which has increased the incidence of side effects ¹⁹. Drug-related side effects often result in a change in treatment, which can adversely affect the treatment’s effectiveness. One of the most common side effects of anti-tuberculosis treatment is hepatotoxicity ¹⁸. In the literature, mitochondrial dysfunction and oxidative stress have been reported to be involved in the mechanism of IRC-induced hepatotoxicity ¹⁹. In this study, TPP 10 and CE ¹⁴, both with antioxidative activity, were examined biochemically and histopathologically for their protective effects on IRC-induced hepatotoxicity. Our biochemical analysis revealed that IRC use caused an increase in oxidant (MDA) and inflammatory markers (NF-κB, TNF-α, IL -1β, and IL -6), a decrease in antioxidants (tGSH, SOD, and CAT), and an increase in ALT and AST activities.

As mentioned above, the role of LPO in the pathogenesis of isoniazid-induced oxidative liver injury was demonstrated ⁶. Rifampicin, on the other hand, was reported to exhibit additive and even synergistic hepatotoxicity when used in combination with isoniazid ⁴. The cause of this condition has been attributed to rifampicin stimulating hydrolases and the formation of hepatotoxic reactive metabolites from isoniazid ⁸. As is well known, MDA is an LPO product used as a biomarker of oxidative stress ²⁰. MDA exhibits a toxic effect; additionally, converting ROS into active substances causes cell membrane damage, leading to apoptosis and liver necrosis ²¹. An experimental study has found that four weeks of IRC treatment has increased MDA in rat livers ²². By suppressing the increase in MDA in the group administered TPP in conjunction with IRC, it is evident that TPP displays antioxidant properties. It was suggested in the literature that thiamine interacted with free radicals and hydroperoxides and inhibited LPO ²³. Previously, TPP was also tested in oxidative liver injury induced by propofol 11 and metazimol ²⁴ and was found to inhibit the increase in MDA levels in rat livers, similar to our study. In our study, CE suppressed the increase in MDA levels. However, CE suppressed the increase in MDA levels less than TPP. Moselhy et al. reported that aqueous and ethanol extracts of cinnamon could prevent the increase in MDA levels by reducing LPO in carbon tetrachloride-induced oxidative liver injury in rats ²⁵.

The liver tissues of the animals were also analyzed in terms of antioxidants. For this purpose, tissue tGSH levels and SOD and CAT activities were determined. GSH represented the most important endogenous non-enzymatic antioxidant. GSH plays a role in the protection of cells from ROS.10. The literature reported that hydrazine reacted with the sulfhydryl group of GSH, depleting its levels in hepatocytes and causing cell death ²⁶. In our study, tGSH levels were found to be decreased in the IRC group. Similarly, Zhang et al. found that IRC decreased GSH levels in liver tissue and serum samples ²⁷. During the metabolism of antituberculosis drugs, toxic metabolites and free radicals deplete endogenous antioxidants, making the liver susceptible to further damage ²⁸. According to the literature, external administration of agents with antioxidant activity could contribute to tissue defense in such cases ²⁹. Our study determined that TPP administered with IRC prevented the decrease in tGSH levels and even maintained them at healthy levels for the healthy group. Delen et al. similarly concluded that TPP inhibited the depletion of tGSH in the liver caused by propofol use 11. The decrease in tGSH levels in CE-treated animals was suppressed, although not as much as in TPP-treated animals. In a previous study, CE was found to possess antioxidant properties and to suppress GSH depletion in liver tissue ²⁹.

Results of our analysis indicated that SOD and CAT activities were also decreased in the IRC group in which tGSH levels were low. By accelerating the conversion of oxygen to hydrogen peroxide (H₂O₂), SOD was the first antioxidant enzyme to fight ROS ²⁷. CAT, on the other hand, converted the toxic H₂O₂ into H₂O and oxygen ³⁰. As a result, SOD and CAT acted as mutually supportive antioxidant enzymes that protected ROS ²⁷. It was demonstrated in previous preclinical studies that IRC decreased the activity of SOD and CAT in the liver ^31,32. The results of this study revealed that TPP treatment was able to prevent the decline in SOD and CAT activities. Throughout the body, TPP catalyzes various chemical reactions. As a cofactor of enzymes involved in maintaining cell redox, it synthesized reduced nicotinamide adenine dinucleotide phosphate and glutathione and increased the synthesis of antioxidants ²⁴. In addition, Bedir et al. found that TPP prevented metamizole-induced decreases in the activities of SOD and CAT in the liver tissues of rats ²⁴. El-Kholy et al. used CE, another therapeutic drug, to treat oxidative liver damage induced by amoxicillin/clavulanate and found that it inhibited the decrease in SOD and CAT activity, similar to our results ²⁹.

Previously, it was reported that IRC treatment caused inflammation. This leads to activation of macrophages and infiltration of circulating immune cells into the tissue ²⁸. In contrast, sterile inflammation also contributed to hepatotoxicity by increasing oxidative stress. NF-κB was a transcription factor that modulated inflammation ^28,33. According to our biochemical results and previous studies, increased NF-κB levels with IRC treatment confirmed inflammatory activity ^28,31. In the TPP-treated group, the increase in NF-κB levels seemed to be prevented. Similarly, Ozer et al. found that TPP suppressed the increase of NF-κB in damaged ovarian tissue ³⁴. In our CE-treated group, NF-κB levels were higher than the TPP group and lower than the IRC group. A recent study found that this increase was suppressed in obese rats treated with Cinnamon powder compared to rats with increased hepatic NF-κB expression with obesity ³⁵. Our study also investigated liver tissues for the levels of proinflammatory cytokines such as TNF-α, IL -1β, and IL -6.

Increased ROS released inactive NF-κB through a series of reactions. Activated NF-κB induced gene transcription of TNF-α and IL -1β ²¹. On the other hand, TNF-α, which is considered the primary mediator inducing the inflammatory response, has been reported to activate NF-κB by binding to the TNF receptor ^31,36. In addition, it was pointed out that TNF-α increased ROS production that triggered liver injury through various pathways ³¹. IL -1β had intense proinflammatory activity like TNF-α ³⁷. Although IL -6 also prevented acute liver inflammation, its prolonged stimulation resulted in tissue damage to the liver ³⁸. Our study discovered that TNF-α, IL -1β, and IL -6 levels were similarly increased in the IRC group in which NF-κB levels were increased. Patel et al. also found an increase in TNF-α, IL -1β, and IL -6 levels in their study on the effects of IRC on the liver ³⁸. Cytokine levels in rats administered with TPP and CE were lower than those of the IRC group. No studies have been conducted in the literature on the effect of TPP on tissue cytokine levels in oxidative liver injury. However, it was reported that TPP prevented the increase in TNF-α and IL -1β levels due to ethanol toxicity in optic nerve tissue ³⁹. In the literature, studies also reported that CE, another therapeutic agent, exhibited anti-inflammatory activity by suppressing the increase in TNF-α, IL -1β, and IL -6 levels induced by various causes in the liver ^40,41.

In this study, the ALT and AST activities of blood samples obtained from animals were measured. These transaminases were sensitive markers of liver cell damage, and these biomarkers have been widely used in the past and present to assess liver function ⁴². In both clinical and experimental studies, it has been demonstrated that IRC treatment increases the levels of ALT and AST ^18,26,42. Our study also demonstrated increased ALT and AST activity after IRC treatment. A lower enzyme activity was observed in both TPP and CE-treated groups, but a lower activity was observed in the TPP-treated group. As previously reported, TPP inhibited the increase in ALT, AST, and lactate dehydrogenase activities following acetaminophen ⁴³ and cisplatin-induced liver injury ¹². Mosely et al. also demonstrated that CE inhibited the increase in ALT and AST activities ²⁵.

A histologic analysis of liver tissues confirmed our biochemical findings. Degeneration of hepatocytes and marked congestion of blood vessels were observed in the IRC group. In addition, an increase in the number of Kupffer cells and PMNL was observed. According to previous studies, IRC induced changes in the liver, such as sinusoidal dilatation, infiltration of immune cells, inflammation in the portal system, and necrosis of the liver ^27,28. Our histopathological analysis showed that the hepatocytes and Kupffer cell populations of the TPP-treated group were similar to those of the control group, and rare evidence of PMNL infiltration was observed. Several studies demonstrated that TPP protected liver tissue from oxidative damage and prevented structural changes such as cellular degeneration, necrosis, and infiltration of inflammatory cells ^11,43. In the CE group, there was moderate hepatocyte degeneration and vascular congestion. In addition, there was a moderate increase in the Kupffer cell population and mild PMNL infiltration. However, some previous studies reported that CE treatment altogether ⁴⁴ or almost entirely ²⁵ prevented tissue damage in oxidative liver injury. However, our histopathologic evaluation indicated that TPP protected the histologic structure of the liver better than CE, similar to our biochemical results.