Perindopril Attenuates Diethylnitrosamine-Induced Hepatocellular Carcinoma in Wistar Rats Through Modulation of Oxidative Stress, Inflammatory Cytokines, And Hepatic Injury Biomarkers

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and accounts for approximately 75–85% of all liver cancers. It represents a major global health challenge and remains among the leading causes of cancer-related mortality worldwide.¹˒² Despite advances in diagnosis and treatment, the incidence and mortality of HCC continue to rise due to the increasing prevalence of chronic liver diseases, including viral hepatitis B and C infections, alcohol-induced liver injury, and metabolic dysfunction-associated fatty liver disease (MAFLD).²˒³

The pathogenesis of HCC is a multifactorial process involving chronic inflammation, oxidative stress, fibrosis, angiogenesis, and genomic instability.⁴ Persistent hepatic injury promotes excessive generation of reactive oxygen species (ROS), leading to lipid peroxidation, DNA damage, mitochondrial dysfunction, and activation of various inflammatory signaling pathways.⁵˒⁶ These molecular events contribute to hepatocyte transformation, uncontrolled cellular proliferation, and tumor progression. Furthermore, chronic inflammatory responses within the liver microenvironment facilitate fibrosis and cirrhosis, which are recognized as major predisposing factors for HCC development.⁴˒⁵

Recent evidence suggests that the renin–angiotensin–aldosterone system (RAAS), traditionally known for its role in cardiovascular regulation, also plays a significant role in cancer biology.⁷ Angiotensin II, the principal effector peptide of RAAS, has been shown to stimulate angiogenesis, inflammation, fibrosis, and cellular proliferation through activation of multiple intracellular signaling pathways, including nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and transforming growth factor-beta (TGF-β)-mediated pathways.⁸˒⁹ These effects contribute to tumor growth, invasion, and metastatic potential. Consequently, pharmacological inhibition of RAAS has emerged as a promising strategy for suppressing tumor progression and improving cancer outcomes.⁷˒⁸

Perindopril is a long-acting angiotensin-converting enzyme (ACE) inhibitor widely prescribed for the management of hypertension, heart failure, and other cardiovascular disorders.¹⁰ In addition to its established cardiovascular benefits, Perindopril exhibits several pleiotropic pharmacological effects, including antioxidant, anti-inflammatory, anti-fibrotic, and anti-angiogenic activities.¹¹˒¹² By reducing the formation of angiotensin II and enhancing the bioavailability of protective mediators such as bradykinin, Perindopril may interfere with critical molecular mechanisms involved in hepatocarcinogenesis. These properties suggest that Perindopril could provide therapeutic benefits beyond cardiovascular disease and may represent a potential candidate for drug repurposing in cancer management.¹¹˒¹²

Therefore, the present study was undertaken to evaluate the protective and therapeutic potential of Perindopril against diethylnitrosamine (DEN)-induced hepatocellular carcinoma in Wistar rats through assessment of physiological, biochemical, oxidative stress, inflammatory, and histopathological parameters.

2. MATERIALS AND METHODS

2.1 Chemicals and Reagents

Diethylnitrosamine (DEN) was procured from Sigma-Aldrich (India) and used for induction of hepatocellular carcinoma. Perindopril was obtained from a certified pharmaceutical source and used as the test drug. 5-Fluorouracil (5-FU) was employed as the standard anticancer agent. All chemicals and reagents used in the study were of analytical grade.

2.2 Experimental Animals

Healthy adult male albino Wistar rats weighing 150–200 g and aged 3–4 months were used in the study. Animals were obtained from a CPCSEA-approved breeding facility and housed in polypropylene cages under standard laboratory conditions (25 ± 1°C, 12 h light/dark cycle, relative humidity 50–60%). Animals had free access to standard pellet diet and water ad libitum throughout the experimental period.

All experimental procedures were conducted in accordance with CPCSEA guidelines and were approved by the Institutional Animal Ethics Committee (IAEC).¹³

2.3 Induction of Hepatocellular Carcinoma

Hepatocellular carcinoma was induced by intraperitoneal administration of diethylnitrosamine (DEN) at a dose of 100 mg/kg body weight once weekly for six weeks. Animals were monitored regularly for changes in body weight, behavioral abnormalities, and signs of disease progression.¹⁴˒¹⁵

2.4 Experimental Design

Animals were randomly divided into five groups (n = 6 per group):

Group I (Normal Control): Received 0.25% CMC orally.

Group II (Carcinogen Control): Received DEN (100 mg/kg, i.p.).

Group III (Positive Control): Received DEN along with 5-Fluorouracil (10 mg/kg, i.p.).

Group IV (Perindopril Low Dose): Received DEN along with Perindopril (1 mg/kg/day, p.o.).

Group V (Perindopril High Dose): Received DEN along with Perindopril (10 mg/kg/day, p.o.).

Treatment was continued for 15 days following successful induction of hepatocellular carcinoma.

2.5 Assessment of Physiological Parameters

Body weight of animals was recorded weekly throughout the study. At the end of the experimental period, animals were sacrificed and livers were excised, washed with ice-cold saline, and weighed. Tumor incidence and visible hepatic nodules were recorded.

2.6 Biochemical Analysis

Blood samples were collected and centrifuged to obtain serum. Liver function biomarkers including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were estimated using commercially available diagnostic kits according to the manufacturer’s instructions.²⁴

2.7 Assessment of Oxidative Stress Markers

Liver tissues were homogenized in phosphate buffer (0.1 M, pH 7.4) and centrifuged. The resulting supernatant was used for biochemical estimations. Superoxide dismutase (SOD) activity was measured based on inhibition of epinephrine auto-oxidation.¹⁶ Catalase (CAT) activity was determined by monitoring decomposition of hydrogen peroxide.¹⁷ Reduced glutathione (GSH) levels were estimated using Ellman’s reagent (DTNB).¹⁸ Malondialdehyde (MDA), a marker of lipid peroxidation, was determined using the thiobarbituric acid reactive substances (TBARS) assay.¹⁹ Protein carbonyl content was quantified using the 2,4-dinitrophenylhydrazine (DNPH) method.²⁰

2.8 Estimation of Pigment Markers and Lipid Profile

Conjugated bilirubin and biliverdin levels were determined spectrophotometrically. Serum lipid profile including total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) was analyzed using standard enzymatic methods.²⁴

2.9 Estimation of Inflammatory Cytokines

Levels of inflammatory cytokines including IL-2, IL-6, IL-10, and IL-1β were quantified using commercially available ELISA kits according to the manufacturer’s protocol. Cytokine concentrations were calculated using standard calibration curves.²¹

2.10 Histopathological Examination

Liver tissues were fixed in 10% neutral buffered formalin, processed through graded alcohols, cleared in xylene, and embedded in paraffin wax. Sections of 4–5 μm thickness were prepared using a rotary microtome and stained with hematoxylin and eosin (H&E). Histological examination was performed under a light microscope to assess hepatocellular degeneration, necrosis, inflammatory infiltration, fibrosis, and neoplastic changes.²²

2.11 Statistical Analysis

Data were expressed as mean ± SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. A value of p < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism software.

RESULTS

3.1 Effect of Perindopril on Body Weight, Liver Weight, and Tumor Incidence

DEN administration produced a marked reduction in body weight and significantly increased liver weight and tumor incidence compared with the normal control group. Perindopril treatment attenuated DEN-induced body weight loss and reduced tumor burden in a dose-dependent manner. The higher dose (10 mg/kg) demonstrated greater efficacy, improving body weight gain (14 ± 1 g) and reducing tumor incidence (10.00 ± 1.97) compared with the low-dose group (1 mg/kg). These findings indicate a protective effect of Perindopril against DEN-induced hepatocarcinogenesis.

Table 1. Effect of Perindopril on Physiological Parameters (Body Weight Variation, Liver Weight, and Tumor Incidence) in DEN-Induced HCC in Albino Wistar Rats.

Sr. No.	Groups	Weight variation (Mean ± SD) (g)	Liver weight (Mean ± SD) (g)	Tumor Incidence (No.)
1	NC	23 ± 1 g	2.81 ± 0.73	0.00 ± 0.00
2	CC	-19 ± 1 g	3.88 ± 0.67	26.00 ± 2.71
3	PC	17 ± 1 g	4.68 ± 0.58	8.00 ± 1.37*
4	T1	9 ± 1 g	6.09 ± 0.56	12.00 ± 2.83*
5	T2	14 ± 1 g	4.45 ± 0.48	10.00 ± 1.97*

 

 

Figure 1. Effect of Perindopril on physiological parameters in DEN-induced HCC in Wistar rats

Table 3. Effect of Perindopril on Serum Enzymatic Biomarkers (ALT, AST, and LDH) in DEN-Induced HCC in Wistar Rats.

Sr. No.	Group	ALT (Mean ± SD) (U/L)	AST (Mean ± SD) (U/L)	LDH (Mean ± SD) (U/L)
1	NC	20.57 ± 1.81	31.13 ± 2.03	15.17 ± 4.54
2	CC	70.14 ± 2.03	83.90 ± 3.10	59.23 ± 5.72
3	PC	45.27 ± 2.85	51.43 ± 6.56	22.34 ± 1.51
4	T1	51.06 ± 4.97	55.35 ± 6.70	47.22 ± 2.52
5	T2	38.96 ± 4.79	45.39 ± 6.70	20.34 ± 1.15

 

 

Figure 2. Effect of Perindopril on serum enzymatic biomarkers (ALT, AST, and LDH) in DEN-induced HCC in Wistar rats.

 

 

Figure 3. Effect of Perindopril on hepatic antioxidant enzyme levels in DEN-induced HCC in Wistar rats.

Table 5. Effect of Perindopril on Oxidative Damage Markers in DEN-Induced HCC in Wistar Rats. **Values are expressed as Mean ± SD (n = 6). Statistical analysis was performed using one-way ANOVA followed by Bonferroni multiple comparison test. ns = non-significant, *P < 0.05, and *P < 0.001 compared with the CC group. NC: Normal Control; CC: Carcinogen Control; PC: Positive Control (5-FU); T1: Perindopril (1 mg/kg); T2: Perindopril (10 mg/kg).

Sr. No.	Group	TBARS/MDA (nM/µg protein)	Protein Carbonyl (µM/µg protein)
1	NC	27.73 ± 0.50	0.11 ± 0.01
2	CC	48.78 ± 2.69	0.38 ± 0.01
3	PC	32.72 ± 1.29***	0.16 ± 0.04***
4	T1	45.13 ± 4.01ns	0.29 ± 0.08*
5	T2	30.97 ± 3.90***	0.19 ± 0.02***

 

 

Figure 7.5. Effect of Perindopril on hepatic oxidative stress markers in DEN-induced HCC in Wistar rats

 

 

Figure 7.6. Effect of Perindopril on hepatic pigment markers in DEN-induced HCC in Wistar rats.

 

 

Figure …. Effect of Perindopril on serum lipid profile parameters in DEN-induced HCC in Wistar rats.

 

 

Figure 7.12. Effect of Perindopril on hepatic inflammatory cytokine levels (IL-2, IL-6, IL-10, and IL-1β) in DEN-induced HCC in Wistar rats.

 

 

Figure 6. Gross morphological evaluation of liver tissues in experimental groups.

 

 

Figure 7. Histopathological evaluation of liver tissues (H&E staining, 400×; scale bar = 50 µm).

(A) Normal control liver showing preserved hepatic architecture, intact hepatocytes, normal nuclei (N), and prominent Kupffer cells (K); (B) carcinogen control liver displaying severe architectural distortion, inflammatory infiltration, tumor aggregate cells (Ta), regenerative clusters (RC), degenerated nuclei (dN), and necrotic areas; (C) 5-FU-treated group showing partial restoration of hepatic architecture with reduced tumor burden and preserved hepatocyte morphology; (D) Perindopril-treated group (1 mg/kg) demonstrating improved hepatocyte organization and reduced inflammatory changes; (E) Perindopril-treated group (10 mg/kg) exhibiting near-normal hepatic architecture with intact hepatocytes, well-defined nuclei, prominent Kupffer cells, and minimal degenerative alterations.

DISCUSSION

Hepatocellular carcinoma (HCC) is a multifactorial disease characterized by oxidative stress, chronic inflammation, metabolic disturbances, and progressive deterioration of liver function.⁴˒⁵ In the present study, DEN administration successfully induced HCC, as evidenced by alterations in physiological, biochemical, oxidative stress, inflammatory, and histopathological parameters. Treatment with Perindopril significantly attenuated these changes, suggesting its potential hepatoprotective and chemopreventive activity.

Body weight loss is a common feature of experimental carcinogenesis and is often associated with metabolic dysfunction, reduced nutrient utilization, and tumor-associated cachexia.²³ In the present study, DEN-treated animals showed a marked reduction in body weight along with increased liver weight and tumor burden. These findings indicate successful induction of hepatic tumors and progressive liver injury. Perindopril treatment improved body weight gain and reduced tumor incidence, suggesting its ability to counteract the systemic effects of carcinogenesis and suppress tumor progression.

Serum enzymatic biomarkers such as ALT, AST, and LDH are widely recognized indicators of hepatocellular damage.²⁴ The significant elevation of these enzymes in the carcinogen control group reflects loss of membrane integrity and leakage of intracellular enzymes into the circulation. Perindopril treatment significantly reduced ALT, AST, and LDH levels, indicating stabilization of hepatocyte membranes and preservation of liver function. The higher dose produced greater improvement and showed effects comparable to those observed with the standard drug treatment.

Oxidative stress is considered a major contributor to HCC development.⁵˒⁶ Excessive generation of reactive oxygen species can damage cellular lipids, proteins, and nucleic acids, thereby promoting carcinogenesis. In the present study, DEN administration significantly reduced the activities of antioxidant enzymes such as SOD and CAT and depleted GSH levels, indicating impairment of the endogenous antioxidant defense system. Similar reductions in antioxidant defenses have been reported in DEN-induced hepatocarcinogenesis models.¹⁴˒²⁵ Perindopril treatment restored these antioxidant markers, suggesting enhanced scavenging of reactive oxygen species and improved cellular defense against oxidative injury. Restoration of antioxidant status may have contributed substantially to the protective effects observed in liver tissue.

The increased levels of TBARS/MDA and protein carbonyl content observed in DEN-treated animals further confirmed the presence of severe oxidative damage. Elevated TBARS/MDA levels indicate enhanced lipid peroxidation, whereas increased protein carbonyl content reflects oxidative modification of cellular proteins.¹⁹˒²⁰ Perindopril significantly reduced both markers, indicating protection against oxidative degradation of lipids and proteins. These findings suggest that the antioxidant properties of Perindopril play an important role in preventing cellular injury associated with hepatocarcinogenesis.¹¹˒¹²

Alterations in bilirubin and biliverdin metabolism are commonly associated with hepatic dysfunction and oxidative stress.²⁴ The significant elevation of these pigment markers in the carcinogen control group indicated impaired hepatic metabolic activity and disturbed heme degradation pathways. Perindopril treatment effectively normalized bilirubin and biliverdin levels, suggesting improvement in hepatic functional integrity and restoration of normal metabolic processes.

Liver cancer is frequently associated with abnormalities in lipid metabolism.²⁶˒²⁷ The dyslipidemia observed in DEN-treated animals, characterized by elevated TC, TG, LDL, and VLDL levels along with reduced HDL concentrations, is consistent with impaired hepatic lipid handling during carcinogenesis. Perindopril significantly corrected these abnormalities and restored lipid homeostasis. Improvement in lipid profile may not only reflect better liver function but may also contribute to limiting tumor progression by reducing metabolic disturbances associated with cancer development.

Chronic inflammation is a key driving force in HCC initiation and progression.²⁸˒²⁹ Elevated levels of IL-2, IL-6, IL-10, and IL-1β in the carcinogen control group indicated a pronounced inflammatory response within hepatic tissue. Among these cytokines, IL-6 and IL-1β are known to promote tumor growth, survival, angiogenesis, and cellular proliferation.²⁸˒²⁹ Perindopril treatment significantly reduced the levels of all measured cytokines, demonstrating potent anti-inflammatory activity. Suppression of inflammatory mediators may represent an important mechanism through which Perindopril exerts its protective effects against DEN-induced hepatocarcinogenesis.¹¹˒¹²

The biochemical findings were further supported by gross morphological and histopathological observations. DEN-treated animals exhibited multiple tumor nodules, distorted liver architecture, inflammatory infiltration, degenerative changes, and necrotic lesions. In contrast, Perindopril-treated groups showed marked improvement in liver morphology and histological organization. The high-dose group demonstrated near-normal hepatic architecture with reduced tumor burden and minimal degenerative changes. These observations strongly support the biochemical evidence of hepatoprotection and indicate effective attenuation of carcinogen-induced hepatic injury.

Overall, the findings of the present study suggest that Perindopril exerts significant protective effects against DEN-induced HCC through multiple mechanisms, including enhancement of antioxidant defenses, reduction of oxidative damage, suppression of inflammatory responses, normalization of lipid metabolism, and preservation of hepatic architecture.⁷˒¹¹˒¹² The greater efficacy observed at the higher dose further indicates a dose-dependent protective effect. These results highlight the potential of Perindopril as a promising candidate for further investigation in the prevention and management of hepatocellular carcinoma.

CONCLUSION

The present study demonstrates that Perindopril exerts significant protective effects against DEN-induced hepatocellular carcinoma in Wistar rats. Treatment with Perindopril effectively reduced tumor burden, improved body weight, normalized liver function biomarkers, and restored hepatic architecture. In addition, Perindopril enhanced endogenous antioxidant defenses by increasing SOD, CAT, and GSH levels while reducing lipid peroxidation and protein oxidation, indicating a substantial attenuation of oxidative stress.

Perindopril also improved hepatic metabolic function by normalizing bilirubin, biliverdin, and lipid profile parameters. Furthermore, the marked reduction in pro-inflammatory cytokines, including IL-2, IL-6, IL-10, and IL-1β, highlights its anti-inflammatory and immunomodulatory properties. These biochemical findings were strongly supported by gross morphological and histopathological observations, which revealed significant protection against DEN-induced hepatic damage and neoplastic alterations.

Overall, the findings suggest that the hepatoprotective and chemopreventive effects of Perindopril are mediated through the combined suppression of oxidative stress, inflammation, and metabolic disturbances associated with hepatocarcinogenesis. The higher dose of Perindopril demonstrated superior efficacy throughout the study. These results indicate that Perindopril may represent a promising therapeutic candidate for the prevention and management of hepatocellular carcinoma. However, further mechanistic studies and clinical investigations are required to fully establish its potential role in cancer therapy.

DECLARATIONS

Ethics Approval and Consent to Participate

All animal experimental procedures were performed in accordance with the guidelines of the CPCSEA, Government of India, and were approved by the IAEC Approval No. 1698/PO/Re/S/13/CPCSEA.

Consent for Publication

Not applicable.

Availability of Data and Materials

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Funding

The authors declare that no external funding was received for this study.

Conflict of Interest

The authors declare that they have no competing interests.

Authors' Contributions

Virendra Kumar Baheliya conceived and designed the study, performed the experiments, analyzed the data, and drafted the manuscript. Dharmendra Singh Rajput, Naveen Gupta, Ganesh Prasad Patel, and Brajmohan Kaushal contributed to the experimental work, data analysis, and manuscript review. Ritesh Suresh Bhate supervised the study, validated the methodology, and critically revised the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors express sincere gratitude to the Department of Pharmaceutical Sciences, Faculty of Pharmacy, Madhyanchal Professional University, Bhopal, for providing the necessary facilities and support to carry out this research work. The authors also acknowledge the valuable guidance and encouragement provided by faculty members and research colleagues throughout the study.

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