Abstract
Background and Aim
Hepatotoxicity represents a significant adverse effect associated with cancer treatment. The present study was designed to evaluate the possible hepatoprotective effects of bevacizumab and nivolumab when administered concomitantly with cisplatin.
Materials and Methods
A total of forty-two male Wistar Albino rats were randomly allocated into six groups: control, bevacizumab (10 mg/kg), nivolumab (3 mg/kg), cisplatin (12 mg/kg), cisplatin plus bevacizumab, and cisplatin plus nivolumab. Histological assessment of liver tissues was performed using hematoxylin and eosin (H&E) and Masson’s trichrome staining. Immunohistochemical evaluation was conducted for inflammatory markers (TNF-α, IL-6), the angiogenic factor VEGF, and apoptotic markers (Bax, Bcl-2, Caspase-3).
Results
Administration of cisplatin resulted in hepatotoxic changes, including disruption of normal hepatic cord architecture, cytoplasmic vacuolization, hemorrhage, mononuclear cell infiltration, and enhanced collagen accumulation. Co-treatment with bevacizumab or nivolumab significantly alleviated these histopathological changes (p<0.001). In addition, levels of inflammatory and pro-apoptotic markers (TNF-α, Bax, Caspase-3) were markedly reduced, whereas expression of the anti-apoptotic protein Bcl-2 was increased in the combination treatment groups compared with the cisplatin-only group. In the cisplatin + nivolumab group, the TNF-α level was 1.0 (0.8–1.2), whereas in the cisplatin-only group it was 2.0 (1.8–2.0) (p=0.03). The Bcl-2 level in the cisplatin + nivolumab group was 1.0 (0.8–1.2), while it was 0.2 (0–0.6) in the cisplatin group (p=0.04).
Conclusion
Bevacizumab and nivolumab exhibited hepatoprotective properties when combined with cisplatin, as demonstrated by histological improvement and regulation of inflammatory and apoptotic signaling pathways.
Keywords: Bevacizumab, liver Injury, nivolumab
Introduction
Recent advances in cancer therapy, particularly immune checkpoint inhibitors (ICIs) and targeted agents, have improved clinical outcomes across multiple malignancies.[1,2] Nivolumab, a programmed death-1 (PD-1) monoclonal antibody, and bevacizumab, a vascular endothelial growth factor (VEGF) inhibitor, are widely used alone or in combination with chemotherapy for several cancers, including hepatocellular carcinoma (HCC).[3]
Chemotherapy may enhance the efficacy of immune checkpoint inhibitors by increasing tumor antigen exposure after cancer cell death and by depleting immunosuppressive cells in the tumor microenvironment.[4] Likewise, bevacizumab inhibits angiogenesis and promotes vascular normalization, improving intratumoral chemotherapy delivery.[5]
The liver is an essential organ indispensable for sustaining life, and hepatic metastasis is associated with poor prognosis regardless of the primary tumor site.[6] Moreover, liver metastases are known to induce an immunosuppressive tumor microenvironment.[7] Chemotherapeutic agents can induce various hepatic injuries, including acute hepatitis, hepatic necrosis, portal vein thrombosis, sinusoidal obstruction syndrome, and hepatic steatosis.[8]
Cisplatin, one of the most widely used cytotoxic agents, can induce hepatotoxicity primarily through oxidative stress and mitochondrial dysfunction.[9] Nivolumab may induce hepatotoxicity through T-lymphocyte activation and loss of peripheral tolerance.[10] Conversely, bevacizumab is generally not associated with hepatotoxicity and is even speculated to exert a hepatoprotective effect.
This study sought to characterize the hepatic effects of cisplatin, nivolumab, and bevacizumab individually and in combination, with particular focus on the hepatic impact of cisplatin–nivolumab and cisplatin–bevacizumab regimens.
Materials and Methods
Ethical Procedures and Animals
In this study, a total of forty-two healthy male Wistar Albino rats (8–10 weeks old, weighing 200–270 g) were obtained from the Harran University (HRÜ) Experimental Animal Application and Research Center, Şanlıurfa, Turkey. All animals were housed under controlled environmental conditions (temperature: 22±2°C; relative humidity: 50% ± 10%; light/dark cycle: 12/12 hours). Rats were provided with standard laboratory chow with free access to water. Throughout the experimental period, animal care and handling were performed in accordance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH, USA).
Experimental Design
In this study, forty-two male Wistar Albino rats (8–10 weeks old, weighing 200–270 g) were randomly assigned into six experimental groups as follows:
Control group (n=6): Received a single intraperitoneal (i.p.) injection of normal saline and were sacrificed on day 14.
Bevacizumab (B) group (n=6): Received 10 mg/kg i.p. bevacizumab on days 1 and 7 and were sacrificed on day 14.[11]
Nivolumab (N) group (n=6): Received 3 mg/kg i.p. nivolumab on days 1 and 7 and were sacrificed on day 14.[12]
Cisplatin (C) group (n=8): Received a single i.p. dose of 12 mg/kg cisplatin on day 1 and were sacrificed on day 14.[13]
Cisplatin + Bevacizumab (CB) group (n = 8): Received 12 mg/kg i.p. cisplatin on day 1, followed by bevacizumab 10 mg/kg i.p. on days 1 and 7, and were sacrificed on day 14.
Cisplatin + Nivolumab (CN) group (n = 8): Received 12 mg/kg i.p. cisplatin on day 1, followed by nivolumab 3 mg/kg i.p. on days 1 and 7, and were sacrificed on day 14.
At the end of the experimental period, all animals were sacrificed under deep anesthesia via exsanguination. Liver tissues were collected for histopathological examination under light microscopy.
Histopathological Evaluation
At the end of the experimental period, liver tissue samples from all groups were fixed in 10% neutral buffered formalin for histopathological examination. Following fixation, tissues were dehydrated through a graded series of ethanol, cleared in xylene, and embedded in paraffin to obtain tissue blocks. Sections of 5 µm thickness were cut using a semi-automated microtome (Thermo Shandon Finesse ME+, Runcorn, UK). The obtained sections were stained with hematoxylin and eosin (H&E) for general histological assessment and with Masson’s Trichrome (Trichrome Masson Stain Kit, Sigma-Aldrich, Code: HT15-1KT, St. Louis, MO, USA) for evaluation of collagen deposition and fibrosis.
Histopathological evaluations were performed under a light microscope by an experienced and blinded histologist using a Zeiss Axioskop II microscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany). Images were captured using a Zeiss Axiocam MRc camera system (Carl Zeiss MicroImaging GmbH, Göttingen, Germany) and digitally archived.
Each microscopic sample was semi-quantitatively scored for hepatic degeneration/regeneration according to the following criteria: hepatocellular degeneration, sinusoidal dilatation, inflammatory cell infiltration, hemorrhage, and degree of fibrovascular area formation.[14,15] The severity of lesions was graded on a four-point scale as follows: normal=0, mild=1, moderate=2, and severe=3.
Immunohistochemistry (IHC) Staining
Immunohistochemical analyses were performed on liver tissue sections to evaluate inflammation, apoptosis, and vascular structural alterations. The localization and expression of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and caspase-3 (Cas-3) were examined.
Paraffin-embedded liver tissue blocks obtained from each experimental group were sectioned at 5 µm thickness and deparaffinized. Following deparaffinization, the sections were rinsed and washed in phosphate-buffered saline (PBS) for 5 minutes. Antigen retrieval was performed by boiling the sections in citrate buffer (pH 6.0). After cooling and washing in PBS, endogenous peroxidase activity was blocked using 3% hydrogen peroxide (H2O2) for 10 minutes.
Primary antibodies—TNF-α (Santa Cruz Biotechnology, Inc., Cat. No. sc-52746), IL-6 (Cat. No. sc-28343), VEGF (Cat. No. sc-7269), Bax (Cat. No. sc-7480), Bcl-2 (Cat. No. sc-7382), and Caspase-3 (Cat. No. sc-56053)—were diluted 1:100 and applied to the sections, which were then incubated at +4 °C overnight. After washing, the sections were incubated with a biotinylated secondary antibody using a commercial detection kit (Thermo Scientific, MA, USA; Cat. No. TP-060-HL), according to the manufacturer’s protocol.
3,3′-Diaminobenzidine (DAB) substrate kit (Sigma-Aldrich, St. Louis, MO, USA; Cat. No. D3939) was used as the chromogen. Counterstaining was performed with Mayer’s hematoxylin, and the slides were mounted with Entellan. All sections were examined under a light microscope, and representative images were captured.
For immunohistochemical evaluation, five randomly selected microscopic fields per section were analyzed. Positive immunoreactivity for TNF-α, IL-6, VEGF, Bax, Bcl-2, and Cas-3 was identified as brown cytoplasmic or nuclear staining. Immunohistochemical labeling was evaluated semiquantitatively based on both the intensity and distribution of specific staining. The staining intensity was assessed using the H-score method, where 0 indicated negative staining, 1+ weak, 2+ moderate, and 3+ strong staining. The H-score was calculated according to the following formula:
H-score = (percentage of cells stained at 1+) × 1 + (percentage of cells stained at 2+) × 2 + (percentage of cells stained at 3+) × 3.
This scoring system yields a total value ranging from 0 to 300, with 300 representing 100% of cells showing strong positive staining.[16,17]
Statistical Analysis
All statistical analyses were performed using IBM SPSS Statistics for Windows, version 25.0 (IBM Corp., Armonk, NY, USA). The normality of data distribution was assessed using the Shapiro–Wilk test. Since most variables did not follow a normal distribution, comparisons among groups were conducted using the Kruskal–Wallis test. When statistically significant differences were identified, post hoc pairwise comparisons were performed using the Sidak correction method via JASP software (Version 0.17; University of Amsterdam, The Netherlands). Results are presented as median values and minimum–maximum. A p-value of less than 0.05 was considered statistically significant.
Results
A comparison of body-weight trajectories showed a significant decrease in the cisplatin (C) group relative to controls (244 g vs. 302 g, p=0.001). No other treatment groups exhibited statistically significant differences in body weight (Appendix Table 1).
Table 1.
Histopathological and morphometric parameters in experimental groups
| Groups | Body weight (g) | VC (µm) | Hepatocyte degeneration | Hemorrhage | Sinusoidal dilatation | Inflammatory cell infiltration | FBD |
|---|---|---|---|---|---|---|---|
| Control | 302 (271–317)b | 8.8 (6.7–12.9)b | 0.2 (0–0.4)b | 0.3 (0.2–0.6)b | 0.3 (0–0.6)b | 0.4 (0.2–0.6)b | 0.2 (0.2–0.4)b |
| Bevacizumab | 278 (246–316) | 13.5 (10.2–16.5)b | 0.4 (0.2–0.8)b | 0.4 (0.4–0.8)b | 0.4 (0.2–0.8)b | 0.6 (0.4–0.6)b | 0.4 (0.2–0.6)b |
| Nivolumab | 270 (265–298)b | 13.2 (12–18.5)b | 0.5 (0.2–1)b | 0.6 (0.4–0.8)b | 0.6 (0.4–0.8)b | 0.6 (0.4–0.8)b | 0.4 (0.2–0.6)b |
| Cisplatin | 244 (235–264)a | 86 (60–103)a | 2.1 (1.8–2.6)a | 1.4 (1–1.8)a | 1.4 (1.2–1.6)a | 1.5 (1.2–1.6)a | 1.4 (1.2–1.8)a |
| Cisplatin+ Bevacizumab | 260 (240–276) | 33 (28–39)a,b | 1 (0.8–1.2)a,b | 1 (0.8–1)a | 0.9 (0.8–1)a,b | 0.7 (0.6–1)b | 0.7 (0.4–0.8)b |
| Cisplatin+ Nivolumab | 247 (240–276) | 29 (20–33)a,b | 0.8 (0.6–1)a,b | 1 (0.8–1.2)a | 0.8 (0.6–1)b | 0.6 (0.4–1)b | 0.5 (0.2–0.8)b |
VC: Vascular congestion; FBD: Fibrovascular density. Data are expressed as median (minimum–maximum). a; P<0.05 compared to the control group; b; P<0.05 compared to the cisplatin group.
Histopathologic Examination
Histopathological Findings
Light microscopic examination showed no histological changes in the control, bevacizumab, or nivolumab groups, where hepatocytes were regularly arranged around the central vein and normal lobular and sinusoidal architecture was maintained (Fig. 1a–c).
Figure 1.
Light microscopic micrographs of liver tissue from experimental groups. Control group (a); Bevacizumab group (b); Nivolumab group (c); Cisplatin group (d–f); Cisplatin + Bevacizumab group (g); Cisplatin + Nivolumab group (h). CV: central vein; sinusoid (arrowhead); hepatocyte (arrow); inflammatory cell infiltration (curved arrow); hemorrhage and sinusoidal dilatation (wavy arrow); hepatocellular degeneration (double bracket). (H&E staining; A–C and E–H: x40, D: x20).
In contrast, cisplatin caused clear hepatic injury, including central vein congestion and dilatation, disruption of hepatic cords (Fig. 1d), hepatocellular degeneration with cytoplasmic vacuolation, hemorrhage, and mononuclear cell infiltration (Fig. 1e, f).
Co-administration of bevacizumab or nivolumab with cisplatin (Fig. 1g, h) markedly reduced these effects, with less hepatocellular vacuolation and mononuclear infiltration, and largely preserved central vein and sinusoidal structure.
Body-weight comparison showed a significant reduction in the cisplatin group compared with controls (302 g vs. 244 g, p=0.001), while no significant differences were observed in the other groups (Appendix Table 1).
Masson’s Trichrome Staining Findings
Masson’s Trichrome staining was used to assess connective tissue and collagen deposition in the periportal area. Bevacizumab and nivolumab did not produce any noticeable changes in connective tissue or collagen levels compared with the control group (Appendix Fig.1a–c). In contrast, cisplatin caused a marked increase in collagen deposition and connective tissue, along with distortion of the normal hepatosinusoidal architecture (Appendix Fig. 1d).
Co-administration of bevacizumab or nivolumab with cisplatin reduced these fibrotic changes, showing decreased collagen accumulation and less architectural distortion, with liver morphology appearing similar to the control group (Appendix Fig. 1e, f). Histopathological and morphometric findings are summarized in Table 1.
Immunohistochemical Expression of Inflammatory Markers (TNF-α, IL-6)
To assess hepatic inflammation after treatment with bevacizumab (B), nivolumab (N), cisplatin (C), and their combinations (BC and NC), immunohistochemical staining for TNF-α and IL-6 was performed, and H-scores were calculated for each group.
There was a significant difference in TNF-α expression among the groups (p<0.001). Post hoc analysis showed this difference was mainly due to increased TNF-α in the cisplatin group compared with controls [2.0 (1.8–2.0) vs. 0.6 (0.4–0.8); p=0.02]. TNF-α levels were significantly lower in the cisplatin + bevacizumab [1.0 (0.8–1.2); p=0.03] and cisplatin + nivolumab [1.0 (0.8–1.2); p=0.03] groups than in the cisplatin-only group (Fig. 2).
Figure 2.
Immunohistochemical micrographs of liver sections stained for TNF-α in the experimental groups. Control group (a); Bevacizumab group (b); Nivolumab group (c); Cisplatin group (d); Cisplatin + Bevacizumab group (e); Cisplatin + Nivolumab group (f). TNF-α positive staining (arrowhead); negative staining (arrow). Magnification: x40. Median value was presented for all groups. *Showed statistically difference between cisplatin and other groups. Bevacizumab (B), Nivolumab (N), Cisplatin (C), Cisplatin+Bevacizumab (CB), Cisplatin+Nivolumab (CN).
IL-6 expression also differed significantly (p<0.001), with the greatest increase in the cisplatin group compared with controls [0.4 (0.2–0.8) vs. 1.8 (1.0–2.0); p=0.04]. However, no significant differences were found between cisplatin and the cisplatin + bevacizumab [1.8 (1.0–2.0) vs. 1.1 (0.8–1.2); p=0.05] or cisplatin + nivolumab [1.8 (1.0–2.0) vs. 1.0 (0.6–1.2); p=0.09] groups (Appendix Fig. 2).
Immunohistochemical Expression of VEGF and Apoptosis Markers (Bax, Bcl-2, Caspase-3)
There was a significant difference in VEGF expression among the groups (p<0.001). Subgroup analysis showed that VEGF levels were significantly higher in the cisplatin + bevacizumab group compared with the control group [1.5 (1.2–1.6) vs. 0.7 (0.2–1.0); p=0.04]. VEGF expression was also significantly elevated in the cisplatin + bevacizumab group compared with the cisplatin group [1.5 (1.2–1.6) vs. 0.8 (0.6–1.0); p=0.03] and the bevacizumab group [1.5 (1.2–1.6) vs. 0.7 (0.6–0.8); p=0.03] (Appendix Fig. 3).
Figure 3.
Immunohistochemical micrographs of liver sections stained for Bax in the experimental groups. Control group (a); Bevacizumab group (b); Nivolumab group (c); Cisplatin group (d); Cisplatin + Bevacizumab group (e); Cisplatin + Nivolumab group (f). Bax positive staining (arrowhead); negative staining (arrow). Magnification: x40. *Showed statistically difference between cisplatin and other groups. Bevacizumab (B), Nivolumab (N), Cisplatin (C), Cisplatin+Bevacizumab (CB), Cisplatin+Nivolumab (CN).
Apoptotic activity was assessed by Bax, Bcl-2, and Caspase-3 expression. Bax levels were significantly higher in the cisplatin group compared with controls [2.0 (1.8–2.4) vs. 0.3 (0.2–0.6); p=0.04]. Bax expression was markedly reduced in the cisplatin + bevacizumab [0.8 (0.6–1.0); p=0.04] and cisplatin + nivolumab [0.6 (0.2–0.8); p=0.04] groups compared with cisplatin alone (Fig. 3).
Bcl-2 expression showed the opposite pattern. The cisplatin + bevacizumab group had significantly higher Bcl-2 levels than the cisplatin group [1.1 (0.6–1.4) vs. 0.2 (0–0.6); p=0.04], and the cisplatin + nivolumab group showed a similar increase [1.0 (0.8–1.2) vs. 0.2 (0–0.6); p=0.04] (Fig. 4).
Figure 4.
Immunohistochemical micrographs of liver sections stained for Caspase-3 in the experimental groups. Control group (a); Bevacizumab group (b); Nivolumab group (c); Cisplatin group (d); Cisplatin + Bevacizumab group (e); Cisplatin + Nivolumab group (f). Caspase-3 positive staining (arrowhead); negative staining (arrow). Magnification: x40. *Showed statistically difference between cisplatin and other groups. Bevacizumab (B), Nivolumab (N), Cisplatin (C), Cisplatin+Bevacizumab (CB), Cisplatin+Nivolumab (CN).
Caspase-3 expression also differed significantly. Cisplatin treatment markedly increased Cas-3 compared with controls [2.0 (1.6–2.4) vs. 0.2 (0–0.6); p=0.004]. Both the cisplatin + bevacizumab [1.0 (0.8–1.0); p=0.03] and cisplatin + nivolumab [0.9 (0.4–1.2); p=0.04] groups showed significantly lower Cas-3 levels than the cisplatin group, indicating a protective effect against cisplatin-induced apoptosis (Appendix Fig. 4).
Immunohistochemical expression levels of inflammatory, angiogenic, and apoptotic markers are summarized in Table 2.
Table 2.
Immunohistochemical expression levels of ınflammatory, angiogenic, and apoptotic markers
| Groups | TNF-α | IL-6 | VEGF | Bax | Bcl-2 | Caspase-3 |
|---|---|---|---|---|---|---|
| Control | 0.6 (0.4–0.8)b | 0.4 (0.2–0.8)b | 0.7 (0.2–1) | 0.3 (0.2–0.6)b | 0.5 (0.4–0.6) | 0.2 (0–0.6)b |
| Bevacizumab | 0.6 (0.4–1)b | 0.5 (0.4–0.6)b | 0.7 (0.6–0.8) | 0.3 (0.2–0.4)b | 0.6 (0.4–0.8) | 0.5 (0.2–0.6)b |
| Nivolumab | 0.7 (0.2–0.8)b | 0.5 (0.2–0.8)b | 0.8 (0.6–0.8) | 0.4 (0.2–0.6)b | 0.7 (0.4–1)) | 0.4 (0.2–0.8)b |
| Cisplatin | 2 (1.8–2)a | 1.8 (1–2)a | 0.8 (0.6–1) | 2 (1.8–2.4)a | 0.2 (0–0.6) | 2 (1.6–2.4)a |
| Cisplatin+ Bevacizumab | 1 (0.8–1.2)a,b | 1.1 (0.8–1.2) | 1.5 (1.2–1.6)a,b | 0.8 (0.6–1)b | 1.1 (0.6–1.4)b | 1 (0.8–1)a,b |
| Cisplatin+ Nivolumab | 1 (0.8–1.2)b | 1 (0.6–1.2) | 1.1 (0.8–1.4) | 0.6 (0.2–0.8)b | 1 (0.8–1.2)a,b | 0.9 (0.4–1.2)b |
Data are expressed as median (minimum–maximum). a; p<0.05 compared to the control group. b; p<0.05 compared to the cisplatin group.
Discussion
Cisplatin remains a cornerstone chemotherapeutic agent across multiple malignancies, yet its clinical utility is frequently restricted by dose-dependent adverse events, among which hepatotoxicity represents a notable limitation. In our study, nivolumab and bevacizumab alone did not cause any histopathological liver changes. Anti–PD-1 agents are known to induce hepatotoxicity in about 2–5% of patients, and nivolumab-related hepatotoxicity has been reported in roughly 1% of melanoma cases.[18,19] This adverse effect, however, usually appears after the fifth week of treatment.[20] Because the animals in our study were sacrificed on day 14, nivolumab-related hepatotoxicity was not detected.
Bevacizumab has been reported to have a protective effect when combined with chemotherapy in patients with colorectal cancer and liver metastases.[21] Animal studies have also shown that presurgical bevacizumab promotes liver regeneration after hepatectomy in rats,[22] and it can reduce hepatic fibrosis and exhibit hepatoprotective activity in experimental fibrosis models.[23]
In our study, bevacizumab administration in healthy rats caused no histopathological changes under light microscopy. Additionally, no significant differences were found in inflammatory, apoptotic, or anti-apoptotic marker expression compared with the control group. These results align with previous findings, indicating that bevacizumab does not produce adverse effects on normal liver histology in healthy rats.
Cisplatin is known for its dose-limiting toxicities, including nephrotoxicity, neurotoxicity, and hepatotoxicity. In this study, cisplatin caused clear hepatic injury—central vein dilatation and congestion, cytoplasmic vacuolation, mononuclear infiltration, and increased collagen deposition. These findings are consistent with previous reports confirming cisplatin-induced hepatotoxicity.[24]
Cisplatin hepatotoxicity is mainly driven by oxidative stress, inflammation, and apoptosis. Earlier studies in rats have shown that cisplatin increased ROS production and elevated TNF-α, while reducing IL-10 and upregulating pro-apoptotic proteins including caspase-3, together with decreased Bcl-2.[13] Similar increases in oxidative markers, caspase-3, and VEGF have also been documented.[25]
Consistent with these reports, our findings showed that cisplatin significantly increased inflammatory cytokines TNF-α and IL-6. Apoptotic evaluation revealed decreased anti-apoptotic Bcl-2 and increased pro-apoptotic Bax, caspase-3, and VEGF expression. Although oxidative markers were not measured, our results support the conclusion that cisplatin induces hepatotoxicity through inflammatory activation and enhanced apoptotic signaling.
The relationship between cisplatin and VEGF expression remains controversial. In our study, cisplatin did not significantly change VEGF levels compared with the control, bevacizumab, or nivolumab groups. One experimental study reported increased VEGF after cisplatin, but they measured the mean staining area and used a lower dose (7.5 mg/kg), which may explain the difference.[25] Another study using a human ovarian cancer xenograft model found decreased VEGF expression after cisplatin, likely due to differences between tumor-bearing animals and the healthy rats used in our study.[26]
Co-administration of bevacizumab with cisplatin reduced cisplatin-induced hepatic damage. The cisplatin + bevacizumab group showed improved hepatic architecture, better regeneration, and restoration of normal sinusoidal and portal structures. Inflammatory and pro-apoptotic markers (TNF-α, Bax, Caspase-3) were decreased, while VEGF and the anti-apoptotic protein Bcl-2 were increased compared with cisplatin alone.
These findings agree with a meta-analysis showing that bevacizumab enhances chemotherapy efficacy while reducing oxaliplatin-induced sinusoidal injury.[27] To our knowledge, this is the first experimental study demonstrating that bevacizumab co-administration mitigates cisplatin-induced hepatotoxicity.
Co-administration of nivolumab with cisplatin led to clear histological improvement of cisplatin-induced hepatotoxicity, including hepatocyte regeneration, reduced cytoplasmic vacuolization, and restoration of normal hepatic cord and portal structures compared with cisplatin alone. Immunohistochemically, the cisplatin + nivolumab group showed lower TNF-α expression, reduced inflammatory infiltration, and decreased Bax and Caspase-3 levels, along with increased Bcl-2 expression.
Previous studies have shown that different agents can reduce cisplatin-induced hepatotoxicity by limiting oxidative damage, apoptosis, and inflammation. For example, the SGLT2 inhibitor dapagliflozin provided hepatoprotection when given with cisplatin through its antioxidant effects.[28] Ganoderma lucidum mushroom extract similarly reduced cisplatin-induced liver injury by decreasing oxidative stress and related apoptosis.[13] Licorice has also been reported to protect against cisplatin-induced hepatic injury by suppressing reactive oxygen species (ROS) generation.[29]
To the best of our knowledge, this is the first study showing that nivolumab co-administration reduces cisplatin-induced hepatotoxicity. Nivolumab decreased TNF-α levels and reduced inflammatory cell infiltration, suggesting a possible anti-inflammatory effect. In addition, the increase in the anti-apoptotic protein Bcl-2 and the decrease in pro-apoptotic Bax and Caspase-3 indicate an anti-apoptotic influence likely linked to its anti-inflammatory activity. Since many agents that improve cisplatin-induced hepatotoxicity act by reducing oxidative stress and enhancing antioxidant defenses, it may be speculated that nivolumab could also exert indirect antioxidant effects contributing to hepatoprotection.
Our study has some limitations. First, the experiments were performed in healthy rats, and the presence of cancer or liver metastases could influence hepatic responses and lead to different outcomes. Second, as this is an experimental animal study, the results cannot be directly extrapolated to humans without additional clinical validation.
Conclusion
In conclusion, cisplatin administration caused clear hepatotoxicity, as shown by histopathological and immunohistochemical findings. Co-administration of bevacizumab or nivolumab significantly reduced these hepatotoxic effects. These results suggest that both agents may offer hepatoprotective benefits when combined with cisplatin, possibly through anti-inflammatory and anti-apoptotic mechanisms. Further studies in tumor-bearing models and clinical settings are needed to confirm these findings.
Footnotes
How to cite this article: Karhan O, Turedi S. Nivolumab and bevacizumab attenuate cisplatin-induced hepatic inflammation and apoptosis in rats. Hepatology Forum 2026; 7(1):45–50.
Online Appendix Link
https://hepatologyforum.org/storage/upload/files/1767602975-appendix-en.pdf
Ethics Committee Approval
The Harran University Clinical Research Ethics Committee granted approval for this study (date: 07.11.2024, number: 2024/006/01-16).
Informed Consent
Written informed consent was obtained from participants.
Conflict of Interest
The authors has no conflicts of interest to declare.
Financial Disclosure
The authors declared that this study has received no financial support.
Use of AI for Writing Assistance
Not declared.
Author Contributions
Concept – OK, ST; Design – OK, ST; Supervision – OK, ST; Findings – OK, ST; Materials – ST; Data Collection and/or Processing – OK, ST; Analysis and/or Interpretation – OK; Literature Search – OK, ST; Writing Manuscript – OK, ST; Critical Review – OK, ST.
Peer-review
Externally peer-reviewed.
References
- 1.Zamani MR, Šácha P. Immune checkpoint inhibitors in cancer therapy: what lies beyond monoclonal antibodies? Med Oncol. 2025;42(7):273. doi: 10.1007/s12032-025-02822-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Garcia J, Hurwitz HI, Sandler AB, Miles D, Coleman RL, Deurloo R, et al. Bevacizumab (Avastin®) in cancer treatment: A review of 15 years of clinical experience and future outlook. Cancer Treat Rev. 2020;86:102017. doi: 10.1016/j.ctrv.2020.102017. [DOI] [PubMed] [Google Scholar]
- 3.Yau T, Galle PR, Decaens T, Sangro B, Qin S, da Fonseca LG, et al. Nivolumab plus ipilimumab versus lenvatinib or sorafenib as first-line treatment for unresectable hepatocellular carcinoma (CheckMate 9DW): an open-label, randomised, phase 3 trial. Lancet. 2025;405(10492):1851–1864. doi: 10.1016/S0140-6736(25)00403-9. [DOI] [PubMed] [Google Scholar]
- 4.Sordo-Bahamonde C, Lorenzo-Herrero S, Gonzalez-Rodriguez AP, Martínez-Pérez A, Rodrigo JP, García-Pedrero JM, et al. Chemo-Immunotherapy: A New Trend in Cancer Treatment. Cancers (Basel) 2023;15(11):2912. doi: 10.3390/cancers15112912. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Cesca M, Morosi L, Berndt A, Fuso Nerini I, Frapolli R, Richter P, et al. Bevacizumab-induced inhibition of angiogenesis promotes a more homogeneous intratumoral distribution of paclitaxel, improving the antitumor response. Mol Cancer Ther. 2016;15(1):125–135. doi: 10.1158/1535-7163.MCT-15-0063. [DOI] [PubMed] [Google Scholar]
- 6.Waninger JJ, Ma VT, Chopra Z, Pearson AN, Green MD. Evaluation of the prognostic role of liver metastases on patient outcomes: Systematic review and meta-analysis. Cancer J. 2023;29(5):279–284. doi: 10.1097/PPO.0000000000000683. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Xu W, Wang Y, Wang N, Liu J, Zhou L, Guo J. Spatial immune remodeling of the liver metastases: discovering the path to antimetastatic therapy. J Immunother Cancer. 2025;13(3):e011002. doi: 10.1136/jitc-2024-011002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Sharma A, Houshyar R, Bhosale P, Choi JI, Gulati R, Lall C. Chemotherapy induced liver abnormalities: an imaging perspective. Clin Mol Hepatol. 2014;20(3):317–326. doi: 10.3350/cmh.2014.20.3.317. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Katanić Stanković JS, Selaković D, Rosić G. Oxidative damage as a fundament of systemic toxicities induced by cisplatin-the crucial limitation or potential therapeutic target? Int J Mol Sci. 2023;24(19):14574. doi: 10.3390/ijms241914574. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Savino A, Rossi A, Fagiuoli S, Invernizzi P, Gerussi A, Viganò M. Hepatotoxicity in cancer immunotherapy: Diagnosis, management, and future perspectives. Cancers (Basel) 2024;17(1):76. doi: 10.3390/cancers17010076. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Semyachkina-Glushkovskaya O, Diduk S, Anna E, Elina D, Artem K, Khorovodov A, et al. Music improves the therapeutic effects of bevacizumab in rats with glioblastoma: Modulation of drug distribution to the brain. Front Oncol. 2022;12:1010188. doi: 10.3389/fonc.2022.1010188. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Güler İ, Kuzucu İ, Baklacı D, Kum RO, Özhamam Uçaryılmaz E, Özcan M. Evaluation of nivolumab for ototoxic effects: An animal study in rats. J Int Adv Otol. 2020;16(2):218–221. doi: 10.5152/iao.2020.7123. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Hassan HM, Al-Wahaibi LH, Elmorsy MA, Mahran YF. Suppression of cisplatin-induced hepatic injury in rats through alarmin high-mobility group box-1 pathway by ganoderma lucidum: Theoretical and experimental study. Drug Des Devel Ther. 2020;14:2335–2353. doi: 10.2147/DDDT.S249093. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Fouad AA, Qutub HO, Al-Melhim WN. Punicalagin alleviates hepatotoxicity in rats challenged with cyclophosphamide. Environ Toxicol Pharmacol. 2016;45:158–162. doi: 10.1016/j.etap.2016.05.031. [DOI] [PubMed] [Google Scholar]
- 15.Turedi S. Protective/preventive effects of quercetin against cyclophosphamide-induced hepatic inflammation, apoptosis and fibrosis in rats. Hepatol Forum. 2023;4(3):135–141. doi: 10.14744/hf.2023.2023.0026. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Abueid L, Uslu Ü, Cumbul A, Velioğlu Öğünç A, Ercan F, Alican İ. Inhibition of 5-lipoxygenase by zileuton in a rat model of myocardial infarction. Anatol J Cardiol. 2017;17(4):269–275. doi: 10.14744/AnatolJCardiol.2016.7248. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Türedi S, Çelik H, Dağlı Ş N, Taşkın S, Şeker U, Deniz M. An examination of the effects of propolis and quercetin in a rat model of streptozotocin-induced diabetic peripheral neuropathy. Curr Issues Mol Biol. 2024;46(3):1955–1974. doi: 10.3390/cimb46030128. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Schneider BJ, Naidoo J, Santomasso BD, Lacchetti C, Adkins S, Anadkat M, et al. Management of immune-related adverse events in patients treated with immune checkpoint inhibitor therapy: ASCO Guideline update. J Clin Oncol. 2021;39(36):4073–4126. doi: 10.1200/JCO.21.01440. [DOI] [PubMed] [Google Scholar]
- 19.Robert C, Long GV, Brady B, Dutriaux C, Maio M, Mortier L, et al. Nivolumab in previously untreated melanoma without BRAF mutation. N Engl J Med. 2015;372(4):320–330. doi: 10.1056/NEJMoa1412082. [DOI] [PubMed] [Google Scholar]
- 20.Weber JS, Hodi FS, Wolchok JD, Topalian SL, Schadendorf D, Larkin J, et al. Safety Profile of nivolumab monotherapy: A pooled analysis of patients with advanced melanoma. J Clin Oncol. 2017;35(7):785–792. doi: 10.1200/JCO.2015.66.1389. [DOI] [PubMed] [Google Scholar]
- 21.Ribero D, Wang H, Donadon M, Zorzi D, Thomas MB, Eng C, et al. Bevacizumab improves pathologic response and protects against hepatic injury in patients treated with oxaliplatin-based chemotherapy for colorectal liver metastases. Cancer. 2007;110(12):2761–2767. doi: 10.1002/cncr.23099. [DOI] [PubMed] [Google Scholar]
- 22.Mori H, Saito YU, Iwahashi S, Ikemoto T, Imura S, Morine Y, et al. Impact of Bevacizumab on Liver Damage After Massive Hepatectomy in Rats. In Vivo. 2019;33(5):1469–1476. doi: 10.21873/invivo.11626. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Huang Y, Feng H, Kan T, Huang B, Zhang M, Li Y, et al. Bevacizumab attenuates hepatic fibrosis in rats by inhibiting activation of hepatic stellate cells. PLoS One. 2013;8(8):e73492. doi: 10.1371/journal.pone.0073492. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Fathy M, Darwish MA, Abdelhamid AM, Alrashedy GM, Othman OA, Naseem M, et al. Kinetin ameliorates cisplatin-induced hepatotoxicity and lymphotoxicity via attenuating oxidative damage, cell apoptosis and inflammation in rats. Biomedicines. 2022;10(7):1620. doi: 10.3390/biomedicines10071620. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.El-Sharouny SH, Rizk A, Rashed LA, Sayed WM, Elmoneam M. Analysis of the therapeutic role of platelet-rich plasma against cisplatin-induced hepatotoxicity in rats: controversy between oxidative and apoptotic markers. Eur J Anat. 2019;23(3):201–213. [Google Scholar]
- 26.Li W, Wan L, Zhai LY, Wang J. Effects of SC-560 in combination with cisplatin or taxol on angiogenesis in human ovarian cancer xenografts. Int J Mol Sci. 2014;15(10):19265–19280. doi: 10.3390/ijms151019265. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Jácome AA, Oliveira FA, Lino F, Lima J. Effect of adding bevacizumab to chemotherapy on pathologic response to preoperative systemic therapy for resectable colorectal liver metastases: A systematic review and meta-analysis. Clin Colorectal Cancer. 2021;20(3):265–272. doi: 10.1016/j.clcc.2021.05.006. [DOI] [PubMed] [Google Scholar]
- 28.Satyam SM, Bairy LK, Rehman A, Farook M, Khan S, Nair AA, et al. Dapagliflozin: A Promising Strategy to Combat Cisplatin-Induced Hepatotoxicity in Wistar Rats. Biology (Basel) 2024;13(9):672. doi: 10.3390/biology13090672. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Li J, Yang X, Lian X, Li B, Ma Q, Yang L, et al. Licorice attenuates cisplatin-induced hepatotoxicity by alleviating endoplasmic reticulum stress and apoptosis. Front Pharmacol. 2025;16:1557125. doi: 10.3389/fphar.2025.1557125. [DOI] [PMC free article] [PubMed] [Google Scholar]