Yanan Chen1, Haifeng Li2, Meifeng Li3, Shubin Niu4, Jiaxin Wang5, Hongwei Shao6, Ting Li7, Hui Wang8. 1. Guangdong Province Key Laboratory of Biotechnology Drug Candidates, School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, China. Electronic address: ynchen1991@163.com. 2. Guangdong Province Key Laboratory of Biotechnology Drug Candidates, School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, China. Electronic address: lihf@gdpu.edu.cn. 3. Guangdong Province Key Laboratory of Biotechnology Drug Candidates, School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, China. Electronic address: meifeng_li@sina.com. 4. School of Biological Medicine, Beijing City University, Beijing 100084, China. Electronic address: niushubin@bcu.edu.cn. 5. Guangdong Province Key Laboratory of Biotechnology Drug Candidates, School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, China. Electronic address: wjxkeepmoving@163.com. 6. Guangdong Province Key Laboratory of Biotechnology Drug Candidates, School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, China. Electronic address: shawhw2000@163.com. 7. State Key Laboratory of Quality Research in Chinese Medicine/Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Macau, China. Electronic address: tli@must.edu.mo. 8. Guangdong Province Key Laboratory of Biotechnology Drug Candidates, School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, China; School of Biological Medicine, Beijing City University, Beijing 100084, China. Electronic address: vera_wh@126.com.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza polysaccharide (SMP) is one of the most important components in the water extract of Salvia miltiorrhiza Bunge, which has been mainly applied for the prevention or treatment of ischemic encephalopathy and cardiac diseases including myocardial infarction and coronary heart diseases in clinical practice. AIM OF THE STUDY: Our object is to investigate the immune regulation effects of SMP, specifically on the proliferation and cytotoxicity of T lymphocytes through MAPK and NF-κB pathway in peripheral blood of cancer patients. MATERIALS AND METHODS: SMP was prepared through refluxing with ethanol, refluxing with water, Sevage treatment and ethanol precipitation. The lymphocytes were obtained from the peripheral blood of cancer patients. The effect of SMP on T lymphocyte proliferation was investigated by cell counting and flow cytometry. The effect of SMP on the proliferation of cancer cell lines A549, hepG2 and HCT116 was examined by MTT assay. The cytotoxic activity of T lymphocytes treated with SMP was detected by Calcein-acetoxymethyl (Calcein-AM) release. The gene expression of IL-4, IL-6, IFN-γ and toll like receptors (TLRs) was detected by semi-quantitative PCR. The protein expression of mitogen activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling pathway were detected by western blotting. To further verify whether SMP functions through the indicated pathways,, T lymphocytes were treated with SMP and an extracellular regulated protein kinase (ERK) inhibitor (U0126), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125) or an inhibitor of NF-κB inhibitor-α (IκBα) (BAY11-7082), respectively. After 24 h co-treatment, the expressions of p-JNK, p-ERK, IκBα, inhibitory kappa B kinase α (IKKα) and inhibitory kappa B kinase β (IKKβ) protein were detected by western blotting, meanwhile cell numbers of T lymphocytes after inhibition were calculated again by cell counter. RESULTS: SMP dose-dependently promoted the proliferation of T lymphocytes of the cancer patients and significantly improved the cytotoxicity of T lymphocytes against cancer cells. However, SMP showed no effect on the proliferation of the tumor cells from the same source. Furthermore, the gene expression of cytokines including IL-4, IL-6 and IFN-γ were also up-regulated. Moreover, SMP enhanced gene expression of TLR1, TLR2 and TLR4; elevated protein expression of p-JNK and p-ERK; increased protein expression of IKKα, and IKKβ and decreased IκBα levels. Meanwhile, knockdown of ERK、JNK or IκBα expression with specific inhibitor significantly depressed the proliferation of T lymphocytes treated with SMP, corroborating the specific regulation effect of SMP on T lymphocytes through MAPK and NF-κB signaling pathways. CONCLUSION: SMP specifically promotes the proliferation and enhances cytotoxicity of T lymphocytes in peripheral blood of cancer patients through activation of TLRs mediated -MAPK and -NF-κB signaling pathways.
ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza polysaccharide (SMP) is one of the most important components in the water extract of Salvia miltiorrhiza Bunge, which has been mainly applied for the prevention or treatment of ischemicencephalopathy and cardiac diseases including myocardial infarction and coronary heart diseases in clinical practice. AIM OF THE STUDY: Our object is to investigate the immune regulation effects of SMP, specifically on the proliferation and cytotoxicity of T lymphocytes through MAPK and NF-κB pathway in peripheral blood of cancerpatients. MATERIALS AND METHODS:SMP was prepared through refluxing with ethanol, refluxing with water, Sevage treatment and ethanol precipitation. The lymphocytes were obtained from the peripheral blood of cancerpatients. The effect of SMP on T lymphocyte proliferation was investigated by cell counting and flow cytometry. The effect of SMP on the proliferation of cancer cell lines A549, hepG2 and HCT116 was examined by MTT assay. The cytotoxic activity of T lymphocytes treated with SMP was detected by Calcein-acetoxymethyl (Calcein-AM) release. The gene expression of IL-4, IL-6, IFN-γ and toll like receptors (TLRs) was detected by semi-quantitative PCR. The protein expression of mitogen activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling pathway were detected by western blotting. To further verify whether SMP functions through the indicated pathways,, T lymphocytes were treated with SMP and an extracellular regulated protein kinase (ERK) inhibitor (U0126), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125) or an inhibitor of NF-κB inhibitor-α (IκBα) (BAY11-7082), respectively. After 24 h co-treatment, the expressions of p-JNK, p-ERK, IκBα, inhibitory kappa B kinase α (IKKα) and inhibitory kappa B kinase β (IKKβ) protein were detected by western blotting, meanwhile cell numbers of T lymphocytes after inhibition were calculated again by cell counter. RESULTS:SMP dose-dependently promoted the proliferation of T lymphocytes of the cancerpatients and significantly improved the cytotoxicity of T lymphocytes against cancer cells. However, SMP showed no effect on the proliferation of the tumor cells from the same source. Furthermore, the gene expression of cytokines including IL-4, IL-6 and IFN-γ were also up-regulated. Moreover, SMP enhanced gene expression of TLR1, TLR2 and TLR4; elevated protein expression of p-JNK and p-ERK; increased protein expression of IKKα, and IKKβ and decreased IκBα levels. Meanwhile, knockdown of ERK、JNK or IκBα expression with specific inhibitor significantly depressed the proliferation of T lymphocytes treated with SMP, corroborating the specific regulation effect of SMP on T lymphocytes through MAPK and NF-κB signaling pathways. CONCLUSION:SMP specifically promotes the proliferation and enhances cytotoxicity of T lymphocytes in peripheral blood of cancerpatients through activation of TLRs mediated -MAPK and -NF-κB signaling pathways.