title: Chinese herb cinobufagin-reduced cancer pain is associated with increased peripheral opioids by invaded CD3/4/8 lymphocytes.
abstract: OBJECTIVE
  To investigate the mechanism of cinobufagin-reduced cancer pain in mouse cancer pain model and in vitro cell co-culture system.
  
  
  METHODS
  Female Kunming mice were randomly divided into 4 groups. One group of animals was set as normal control without any treatment. Other three groups of animals received H22 hepatoma cell inoculation in right hind paw. At day 9 after inoculation, mice in other three groups were injected intraperitoneally once a day for 8 days with the solvent, morphine or cinobufagin, respectively. The pain behavior was recorded daily. On the last day, all mice were sacrificed and xenograft tissues homogenate and plasma levels of β-endorphin (β-END), corticotropin-releasing factor (CRF) and interleukin-1β (IL-1β) were assessed by ELISA assay. Immunohistochemistry was performed to determine the expression of β-END, pro-opiomelanocortin (POMC) and the μ-opioid receptor (μ-OR) in the xenograft tissues. Immunofluorescence was used to localize lymphocytes with expression of CD3+, CD4+ and CD8+ in xenograft tumors and adjacent tissues. Mice splenic lymphocytes and H22 hepatoma carcinoma ascites cells were prepared for co-culture. β-END and CRF were detected in co-culture supernatants. The MTT assay and cytometry were used to assess cell proliferation. RT-PCR was conducted to determine the gene expression of POMC and Cathepsin L (CTSL). Chemotaxis was examined using a transwell-based migration assay.
  
  
  RESULTS
  Compared to the model group, the thermal and mechanical pain thresholds were increased in mice after cinobufagin treatment. The expression of β-END and CRF in the plasma and tumor tissues of cinobufagin group were much higher than that of the model group mice, but the expression of IL-1β in the plasma and tumor tissues was much lower than that in the model group mice. Meanwhile, the expression of β-END, POMC and μ-OR proteins was significantly increased in the xenograft tissues from cinobufagin group. Lymphocyte population of CD3+, CD4+, CD8+ were also elevated in xenograft tumors and adjacent tissues. In the cell co-culture assays, the content of β-END in the supernatant was significantly increased by cinobufagin in a dose-dependent manner. Cinobufagin also largely increased the proliferation of immune cells and inhibited H22 hepatoma carcinoma cell proliferation in single or co-culture cell assays. Gene expression of POMC and CTSL in cinobufagin group was significantly up-regulated comparing to the control group. Finally, cinobufagin addition enhanced the migration of immune cells in transwell assay.
  
  
  CONCLUSIONS
  Cinobufagin-induced local analgesic effect might be associated with increased activity of POMC/β-END/μ-OR pathway released from invaded CD3/4/8 lymphocytes in cancer tissues.
authors.affiliation: Third-Grade Pharmacological Laboratory on Chinese Medicine Approved by State Administration of Traditional Chinese Medicine, China Three Gorges University, Yichang 443002, China.|Department of Pharmacology, China Three Gorges University, Yichang 443002, China.|Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang 443002, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Chen
authors.affiliation: Third-Grade Pharmacological Laboratory on Chinese Medicine Approved by State Administration of Traditional Chinese Medicine, China Three Gorges University, Yichang 443002, China.|College of Medical Science, China Three Gorges University, Yichang 443002, China.
authors.forename: Shenjun
authors.initials: S
authors.lastname: Yuan
authors.affiliation: Third-Grade Pharmacological Laboratory on Chinese Medicine Approved by State Administration of Traditional Chinese Medicine, China Three Gorges University, Yichang 443002, China.|College of Medical Science, China Three Gorges University, Yichang 443002, China.
authors.forename: Xin-Nian
authors.initials: XN
authors.lastname: Wan
authors.affiliation: Third-Grade Pharmacological Laboratory on Chinese Medicine Approved by State Administration of Traditional Chinese Medicine, China Three Gorges University, Yichang 443002, China.|College of Medical Science, China Three Gorges University, Yichang 443002, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Zhan
authors.affiliation: Third-Grade Pharmacological Laboratory on Chinese Medicine Approved by State Administration of Traditional Chinese Medicine, China Three Gorges University, Yichang 443002, China.|College of Medical Science, China Three Gorges University, Yichang 443002, China.
authors.forename: Xue-Qin
authors.initials: XQ
authors.lastname: Yu
authors.affiliation: Third-Grade Pharmacological Laboratory on Chinese Medicine Approved by State Administration of Traditional Chinese Medicine, China Three Gorges University, Yichang 443002, China.|Department of Pharmacology, China Three Gorges University, Yichang 443002, China.
authors.forename: Jian-Hong
authors.initials: JH
authors.lastname: Zeng
authors.affiliation: Third-Grade Pharmacological Laboratory on Chinese Medicine Approved by State Administration of Traditional Chinese Medicine, China Three Gorges University, Yichang 443002, China.|College of Medical Science, China Three Gorges University, Yichang 443002, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Li
authors.affiliation: Third-Grade Pharmacological Laboratory on Chinese Medicine Approved by State Administration of Traditional Chinese Medicine, China Three Gorges University, Yichang 443002, China.|College of Medical Science, China Three Gorges University, Yichang 443002, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Third-Grade Pharmacological Laboratory on Chinese Medicine Approved by State Administration of Traditional Chinese Medicine, China Three Gorges University, Yichang 443002, China.|College of Medical Science, China Three Gorges University, Yichang 443002, China.
authors.forename: Xiang-Yang
authors.initials: XY
authors.lastname: Hu
authors.affiliation: Third-Grade Pharmacological Laboratory on Chinese Medicine Approved by State Administration of Traditional Chinese Medicine, China Three Gorges University, Yichang 443002, China.|College of Medical Science, China Three Gorges University, Yichang 443002, China.
authors.forename: Yi-Fei
authors.initials: YF
authors.lastname: Ye
authors.affiliation: Third-Grade Pharmacological Laboratory on Chinese Medicine Approved by State Administration of Traditional Chinese Medicine, China Three Gorges University, Yichang 443002, China.|Department of Pharmacology, China Three Gorges University, Yichang 443002, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Hu
mesh_terms: D000700:Analgesics / Q000494:pharmacology*; D000818:Animals; D002018:Bufanolides / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000150:complications; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism; D004195:Disease Models, Animal; D004365:Drugs, Chinese Herbal / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007150:Immunohistochemistry; D053583:Interleukin-1beta / Q000378:metabolism; D008113:Liver Neoplasms / Q000150:complications; D016246:Lymphocytes, Tumor-Infiltrating / Q000378:metabolism; D051379:Mice; D009374:Neoplasms, Experimental / Q000150:complications*; D010146:Pain / Q000188:drug therapy* / Q000209:etiology*; D017288:Pain Threshold; D016133:Polymerase Chain Reaction; D011897:Random Allocation; D001615:beta-Endorphin / Q000378:metabolism
chemical_list: D000700:Analgesics; D002018:Bufanolides; D004365:Drugs, Chinese Herbal; D053583:Interleukin-1beta; D001615:beta-Endorphin; D003346:Corticotropin-Releasing Hormone; C002471:cinobufagin
keywords: cancer pain; cinobufagin; lymphocytes; peripheral opioid receptor; β-endorphin