Korean J Physiol Pharmacol.  2019 Nov;23(6):501-508. 10.4196/kjpp.2019.23.6.501.

Effects of rosmarinic acid on immunoregulatory activity and hepatocellular carcinoma cell apoptosis in H22 tumor-bearing mice

• Cao W
• Mo K
• Wei S
• Lan X
• Zhang W
• Jiang W

• KMID: 2461043
• DOI: http://doi.org/10.4196/kjpp.2019.23.6.501

Abstract

Rosmarinic acid (RA) is a natural polyphenolic compound that exists in many medicinal species of Boraginaceae and Lamiaceae. The previous studies have revealed that RA had therapeutic effects on hepatocellular carcinoma (HCC) in the H22-xenograft models by inhibiting the inflammatory cytokines and NF-κB p65 pathway in the tumor microenvironment. However, its molecular mechanisms of immunoregulation and pro-apoptotic effect in HCC have not been fully explored. In the present study, RA at 75, 150, and 300 mg/kg was given to H22 tumor-bearing mice via gavage once a day for 10 days. The results showed that RA can effectively inhibit the tumor growth through regulating the ratio of CD4⁺/CD8⁺ and the secretion of interleukin (IL)-2 and interferon-γ, inhibiting the expressions of IL-6, IL-10 and signal transducer and activator of transcription 3, thereby up-regulating Bax and Caspase-3 and down-regulating Bcl-2. The underlying mechanisms involved regulation of immune response and induction of HCC cell apoptosis. These results may provide a more comprehensive perspective to clarify the anti-tumor mechanism of RA in HCC.

Keyword

Apoptosis; Hepatocellular carcinoma; Immunoregulation; Rosmarinic acid

MeSH Terms

Animals
Apoptosis*
Boraginaceae
Carcinoma, Hepatocellular*
Caspase 3
Cytokines
Interleukin-10
Interleukin-6
Interleukins
Lamiaceae
Mice*
STAT3 Transcription Factor
Therapeutic Uses
Tumor Microenvironment
Caspase 3
Cytokines
Interleukin-10
Interleukin-6
Interleukins
STAT3 Transcription Factor
Therapeutic Uses

Figure

• 

  Fig. 1 Effects of rosmarinic acid (RA) on CD3+, CD4+, and CD8+ in tumor-bearing mice.(A, C) Percentage of CD3+ T lymphocyte. (B, D, E) Percentages of CD4+ T and CD8+ T lymphocyte. (F) Ratio of CD4+/CD8+. Percentages of CD3+, CD4+, and CD8+ T lymphocytes were determined by flow cytometry. Data were presented as mean ± standard deviation. #p < 0.05 compared with non-tumor group, *p < 0.05 compared with model group.

• 

  Fig. 2 Effects of rosmarinic acid (RA) on cytokine levels in xenograft tumor tissues.tumor tissues were collected after RA treatment. Levels of interleukin (IL)-2 (A), interferon (IFN)-γ (B), IL-6 (C) and IL-10 (D) in tumor tissues were measured by ELISA. Data were presented as mean ± standard deviation. *p < 0.05 compared with model group.

• 

  Fig. 3 Effects of rosmarinic acid (RA) on signal transducer and activator of transcription 3 (STAT3) in xenograft tumor tissues.(A, B) At the end of RA treatment, STAT3 protein expression was measured by immunohistochemistry (magnification, ×400). The images were analyzed by an Image Pro Plus 6.0 system. (C) Total RNA was extracted from xenograft tumor tissues. The expression of STAT3 mRNA was measured by qRT-PCR. GAPDH was used as a loading control. Data were presented as mean ± standard deviation. IOD, integrated opticdensity. *p < 0.05 compared with model group.

• 

  Fig. 4 Effects of rosmarinic acid (RA) on expressions of Bax, Bcl-2, and Caspase-3 in xenograft tumor tissues.Expressions of Bax, Bcl-2, and Caspase-3 proteins were measured by immunohistochemistry (magnification, ×400). The images were analyzed by an Image Pro Plus 6.0 system. Expressions of Bax, Bcl-2, and Caspase-3 mRNA were measured by qRT-PCR. GAPDH was used as a loading control. (A, D) Bax protein expression; (B, E) Bcl-2 protein expression; (C, F) Caspase-3 protein expression. (G) Bax mRNA expression; (H) Bcl-2 mRNA expression; (I) Caspase-3 mRNA expression. Data were presented as mean ± standard deviation. IOD, integrated opticdensity. *p < 0.05 compared with model group.

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