CCl4 damages cells in a variety of organs, primarily the liver, kidneys, and lungs (Teschke, 2018). The production of the CCl3 free radical and other metabolites causes CCl4 poisoning which harms cells through lipid peroxidation and other mechanisms. These free radicals may cause organ failure on several levels (Manno, Rezzadore, Grossi, & Sbrana, 1996). Our experiments were aimed to assess the efficacy of cinnamon extract in modulating the kidney damage caused by CCl4 by monitoring oxidative stress and inflammatory markers as well as performing histological analyses.
Our study involved four research stages in attempt to reach this research goal. The initial stage was studying the phytochemical constituents of CE and determining its antioxidant activity. DPPH, an unchanging nitrogen-centered violet coloured free radical, was used to assess the free radical scavenging potency. This assay relies on the reduction of an alcoholic DPPH solution to non-radical yellow-colored diphyenylpicrylhydrazine. As a result, the radical scavenging effectiveness of plant extracts is determined by measuring a drop in DPPH absorbance caused by colour variation. The strong antioxidant properties of CE was attributed mainly to the presense of terpenoid compounds, as cinnamaldehyde. Further, the polyphenolic compounds present in CE may also contribute to this antioxidant activity. The phenolic group can accept an electron and forming relatively stable phenoxyl radicals which disrupt the chain oxidation reaction (Pandey & Rizvi, 2009).
The second stage of our work was investigating the oxidative stress alterations caused by individual and combined treatment with CCl4 and CE. The oxidative stress biomarkers were examined. When the pro-oxidant/antioxidant equilibrium is altered toward the pro-oxidants, oxidative stress occurs. The presence of polyunsaturated fatty acids in cellular membranes causes oxidative damage, and lipid perioxidation is a useful marker for studying this. MDA is a lipid perioxidation end product that binds to the protein's sulfhydryl groups and has the capacity to crosslink the protein, decreasing or eliminating it (Ayala, Muñoz, & Argüelles, 2014). When compared to the control group, CCl4 caused oxidative stress, as demonstrated by a significant rise in kidney homogenate MDA and a significant drop in kidney homogenate GSH and SOD levels.
Increased MDA levels are linked to a decrease of membrane fluidity and the antioxidant defence systems' inability to scavenge free radicals. All of these variables contribute to tissue damage (Al-Olayan, El-Khadragy, Metwally, & Moneim, 2014). Antioxidant enzymes like SOD and GSH help to protect the body from reactive oxygen species (ROS) and environmental damage. Glutathione acts as a primary line of defence against ROS. Furthermore, GSH-dependent enzymes provide an important line of defence by detoxifying the toxic byproducts produced by ROS. Increased GSH use against peroxides could explain the lower GSH levels in the kidney. Many investigations have shown that GSH plays a critical role in the elimination of CCl4's toxic metabolites, and that organ harm begins when GSH levels are severely reduced (Boshy et al., 2017). These finding agreed with Bellassoued et al. (2019) who demonstrated that giving rats cinnamon essential oil had a great potential for quenching free radicals and alleviating CCl4-induced hepatorenal damage.
As highlighted in our study, CE significantly reduced MDA levels while considerably increasing GSH and SOD levels in CCl4-intoxicated groups, reversing oxidative stress. These findings indicated that CE has a strong antioxidant properties that reduced oxidative stress and helped to restore damaged cells. These findings illustrated that CE's antiradical scavenging is one of the most important mechanisms for preventing cellular damage.
Monitoring the measures of inflammation and kidney damage generated by individual and combined therapy with CCl4 and CE was the third stage of our research. TNF-α is an inflammatory cytokine that is released during acute inflammation and is involved in a variety of signalling pathways within cells that result in necrosis or apoptosis. This protein is also valuable for infection and cancer resistance (Idriss & Naismith, 2000). TNF- levels in CCl4-treated rats' renal tissues homogenate are much higher than in normal control rats. These findings supported Mazani et al. (2020) whose findings revealed that CCL4 injection causes the liver and kidney tissues to produce pro-inflammatory cytokines including IL-6 and TNF-α.
Inhibiting inflammation is a crucial step in reducing CCl4's damage. Our results showed that CE can alleviate against CCl4-induced nephrotoxicity as evidenced by significant decrease in TNF-α concentration in rats’ kidney homogenate. Our findings are agreed with earlier studies, where the expression level of TNF-α was significantly reduced by polyphenolic compounds administration due to their anti-inflammatory activity (Yahfoufi, Alsadi, Jambi, & Matar, 2018).
Kidney Injury Molecule (KIM-1) aids in the phagocytic clearance of apoptotic and necrotic cells during renal injury (Han, Bailly, Abichandani, Thadhani, & Bonventre, 2002). Based on our knowledge, no research has been published on the effect of CE treatment on KIM-1 expression. Compared to normal rats, KIM-1 level in kidney tissue homogenates of CCl4 treated rats are considerably higher. These finding are in the same line with Potić et al. (2019) who reported that CCl4 promotes parenchymatous degeneration in S1/S2 segments of proximal tubules, implying that KIM-1 expression will rise in those regions. Tubulocytes that express KIM-1 are able to phagocyte luminal apoptotic debris, allowing renal tissue to recover and the inflammatory response to be reduced (Zuk & Bonventre, 2016). Autophagy is being considered as a potential pharmacological target for treating some kidney problems as it can allow severely damaged cells to remain and cause harm (Baisantry et al., 2016).
CE significantly decreased KIM-1 level in renal tissue homogenates of CCl4 treated rats compared to rats treated with CCl4 only. The ameliorative effect of CE could be attributed to its flavonoid content. (Ren et al., 2020) found that flavonoid compound inhibited induced mRNA level of KIM-1.
The change in the concentration of the conventional biochemical marker β2-MG was used to identify the indication of kidney injury (Popović et al., 2019). Almost all nucleated cells have β2-MG on their membrane surface. Increased levels of β2-MG have been linked to a variety of illnesses, including inflammation, hepatic or renal disorders, viral infections, and various cancers. (Li, Dong, & Wang, 2016).
In comparison to normal rats, CCl4 treatment resulted in a considerable rise in β2-MG levels in renal tissue homogenates. In comparison to CCl4-treated rats, CE dramatically reduced β2-MG levels in kidney tissue homogenates. The ameliorative effect of CE is attributed mainly to its high content of cinnamaldehyde which results in decreased expression of β2-MG mRNA (El Ali, Deloménie, Botton, Pallardy, & Kerdine-Römer, 2017).
The fourth stage of our research looked at the renal histological structure after treatment with CCl4 and CE separately and in combination. CE treatment reduced CCl4 damage in the kidneys, according to histological evaluations. CE's antioxidant properties helped to reduce oxidative kidney damage.