title: Combination use of ferulic acid, ligustrazine and tetrahydropalmatine inhibits the growth of ectopic endometrial tissue: a multi-target therapy for endometriosis rats.
abstract: BACKGROUND
  Ferulic acid (FA), ligustrazine (LZ) and tetrahydropalmatine (THP) are separately isolated from Chinese Angelica, Szechwan Lovage Rhizome and Rhizoma in the Jiawei-Foshou-San formula, a popular traditional Chinese medicine for irregular menses. It has been reported that the combination use of FA+LZ+THP has similar effect on endometriosis, but the underlying mechanisms are unclear. This study was to investigate the combination effects and mechanisms of FA+LZ+THP on endometriosis rats.
  
  
  METHODS
  Fifty endometriosis rats were intragastricly treated with FA+LZ+THP for 4 wk. The volume of ectopic endometrial tissue was measured to evaluate the effects. Then the changes in hypothalamic-pituitary-ovarian axis and ERE pathway were indicated by the levels of E2, GnRH, FSH and LH, and the expressions of ER, HSP90 and COX-2, respectively. In addition, peritoneal macrophages of each rat were cultured in vitro and treated with (FA+LZ+THP)-medicated serum for 24h. The proliferation and phagocytosis abilities, the levels of IL-1β and TNF-α, and the expression of IκBα were then measured for the changes of peritoneal macrophage activities.
  
  
  RESULTS
  Combination use of FA+LZ+THP diminished the volume of the ectopic endometrial tissues (P<0.05 or P<0.01). It also decreased the E2 level, suppressed the expression of GnRH, FSH and LH, and decreased the protein expression of ER, HSP90 and COX-2 (all P<0.05 or P<0.01). The phagocytosis ability of peritoneal macrophage was enhanced by (FA+LZ+THP)-medicated serum (P<0.05) with no change of proliferation (P>0.05). Moreover, IL-1β and TNF-α were downregulated (both P<0.05 or P<0.01) and IκBα was upregulated by the (FA+LZ+THP)-medicated serum (P<0.01).
  
  
  CONCLUSIONS
  The combination use of FA, LZ and THP could inhibit the growth of ectopic endometrial tissue in endometriosis rats. It might be related to the down-regulation of hypothalamic-pituitary-ovarian axis, the amelioration in ERE pathway and the improvement of peritoneal macrophage activities.
authors.affiliation: College of Pharmaceutical Sciences & College of Traditional Chinese Medicine, Southwest University, Chongqing 400715, China; Institute of Chinese Medicine, Southwest University, Chongqing 400715, China; Pharmacology of Chinese Materia Medica, The Key Constructing Discipline by the State Administrative Bureau of Traditional Chinese Medicine, Chongqing 400715, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Tang
authors.affiliation: College of Pharmaceutical Sciences & College of Traditional Chinese Medicine, Southwest University, Chongqing 400715, China; Institute of Chinese Medicine, Southwest University, Chongqing 400715, China; Pharmacology of Chinese Materia Medica, The Key Constructing Discipline by the State Administrative Bureau of Traditional Chinese Medicine, Chongqing 400715, China.
authors.forename: Fanghong
authors.initials: F
authors.lastname: Shang
authors.affiliation: College of Pharmaceutical Sciences & College of Traditional Chinese Medicine, Southwest University, Chongqing 400715, China; Institute of Chinese Medicine, Southwest University, Chongqing 400715, China; Pharmacology of Chinese Materia Medica, The Key Constructing Discipline by the State Administrative Bureau of Traditional Chinese Medicine, Chongqing 400715, China.
authors.forename: Xiaocui
authors.initials: X
authors.lastname: Wang
authors.affiliation: College of Pharmaceutical Sciences & College of Traditional Chinese Medicine, Southwest University, Chongqing 400715, China; Institute of Chinese Medicine, Southwest University, Chongqing 400715, China; Pharmacology of Chinese Materia Medica, The Key Constructing Discipline by the State Administrative Bureau of Traditional Chinese Medicine, Chongqing 400715, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Research Center of Medical Chemistry and Chemical Biology, Chongqing Technology and Business University, Chongqing 400067, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Chen
authors.affiliation: College of Pharmaceutical Sciences & College of Traditional Chinese Medicine, Southwest University, Chongqing 400715, China; Institute of Chinese Medicine, Southwest University, Chongqing 400715, China; Pharmacology of Chinese Materia Medica, The Key Constructing Discipline by the State Administrative Bureau of Traditional Chinese Medicine, Chongqing 400715, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Chen
authors.affiliation: College of Pharmaceutical Sciences & College of Traditional Chinese Medicine, Southwest University, Chongqing 400715, China; Institute of Chinese Medicine, Southwest University, Chongqing 400715, China; Pharmacology of Chinese Materia Medica, The Key Constructing Discipline by the State Administrative Bureau of Traditional Chinese Medicine, Chongqing 400715, China.
authors.forename: Jifen
authors.initials: J
authors.lastname: Zhang
authors.affiliation: College of Pharmaceutical Sciences & College of Traditional Chinese Medicine, Southwest University, Chongqing 400715, China; Institute of Chinese Medicine, Southwest University, Chongqing 400715, China; Pharmacology of Chinese Materia Medica, The Key Constructing Discipline by the State Administrative Bureau of Traditional Chinese Medicine, Chongqing 400715, China. Electronic address: xuxiaoyu@swu.edu.cn.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Xu
mesh_terms: D000818:Animals; D001600:Berberine Alkaloids / Q000627:therapeutic use*; D002828:Choristoma / Q000188:drug therapy / Q000378:metabolism; D003373:Coumaric Acids / Q000627:therapeutic use*; D004359:Drug Therapy, Combination; D004715:Endometriosis / Q000188:drug therapy* / Q000378:metabolism; D004717:Endometrium / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D004958:Estradiol / Q000097:blood; D005260:Female; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism; D020672:I-kappa B Proteins / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D007986:Luteinizing Hormone / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000502:physiology; D000072000:NF-KappaB Inhibitor alpha; D010587:Phagocytosis / Q000187:drug effects; D011719:Pyrazines / Q000627:therapeutic use*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D001600:Berberine Alkaloids; D003373:Coumaric Acids; D020672:I-kappa B Proteins; D053583:Interleukin-1beta; C000606206:Nfkbia protein, rat; D011719:Pyrazines; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D007987:Gonadotropin-Releasing Hormone; C014215:tetrahydropalmatine; D004958:Estradiol; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; C004999:ferulic acid; C017953:tetramethylpyrazine
keywords: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; An internal control used in Western Blot; CLIA; CMC-Na; COX-2; Carboxymethycellulose (PubChem CID: 6328154); Chemiluminescent Immunoassay; Cycloxygenase-2; DAB; Diaminobenzidine; Diaminobenzidine (PubChem CID: 7071); E(2); ECL; ELISA; ER; ERE; Endometriosis; Enhanced chemiluminescence; Enzyme linked immunosorbent assay; Estradiol; Estradiol (PubChem CID: 5757); Estrogen receptor; Estrogen response element; FA; FBS; FSH; Ferulic acid; Ferulic acid (PubChem CID: 445858); Fetal bovine serum; Follicle-stimulating hormone; GTN; Gestrinone; Gestrinone (PubChem CID: 27812); GnRH; Gonadotropin-releasing hormone; HRP; HSP90; Heat-shock protein 90; Horseradish peroxidase; IL-1β; IgG; Immunoglobulin G; Inhibitor of nuclear factor-κB; Interleukin-1β; IκBα; LH; LPS; LZ; Ligustrazine; Ligustrazine (PubChem CID: 14296); Lipopolysaccharide; Lipopolysaccharide (PubChem CID: 11970143); Luteinizing hormone; MTT; NF-κB; NP-40; Nonyl phenoxypolyethoxylethanol; Nuclear transcription factor-κB; PAGE; PBS; PMSF; PVDF; Phenylmethanesulfonyl fluoride; Phosphate buffered saline; Polyacrylamide gel electrophoresis; Polyvinylidene difluoride; RIPA; RPMI; Radio immunoprecipitation assay; Rats; Roswell Park memorial institute; SDS; SP; Sodium Carboxymethycellulose; Sodium dodecyl sulfate; Sodium dodecyl sulfate (PubChem CID: 3423265); Streptavidin–peroxidase; TBST; THP; TNF-α; Tetrahydropalmatine; Tetrahydropalmatine (PubChem CID: 72301); Tris-buffered saline and Tween 20; Tumor necrosis factor; β-actin