Liuwei Dihuang Pill(六味地黄丸)Treats Postmenopausal Osteoporosis with Shen(Kidney) Yin Deficiency via Janus Kinase/Signal Transducer and Activator of Transcription Signal Pathway by Up-regulating Cardiotrophin-Like Cytokine Factor 1 Expression
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摘要
Objectives: To investigate the mechanism of Liuwei Dihuang Pill(六味地黄丸, LDP) in treating postmenopausal osteoporosis(PMOP) with Shen(Kidney) yin deficiency. Methods: In this study, 205 cases of PMOP were divided into the PMOP Shen-yin deficiency group(Group A), PMOP Shen-yang deficiency group(Group B), PMOP without Shen deficiency group(Group C), and control group(Group N). Real-time polymerase chain reaction(RT-PCR) and Western blot techniques were used to observe the effects of LDP treatment on the cardiotrophin-like cytokine factor 1(CLCF1), ankyrin repeat and SOCS box containing 1(ASB1), and prokineticin 2(PROK2) genes and the Janus kinase/signal transducer and activator of transcription(JAK/STAT) signaling pathway. Results: The m RNA(P<0.05) and protein(P<0.01) expression levels of the CLCF1 gene in Group A were significantly lower than the corresponding levels in Group N. After LDP treatment for 3 months, the m RNA expression levels of the CLCF1 gene were obviously up-regulated(P<0.01). After 6-month treatment, the expression levels of CLCF1 m RNA and protein were significantly up-regulated(both P<0.01), and the average bone density of the top femur had significantly increased(P<0.05). In vitro, CLCF1 overexpression resulted in a significant increase in the total protein and phosphorylated protein levels of JAK2 and STAT3. Conclusions: The CLCF1 gene is an important gene associated with PMOP Shen-yin deficiency and the therapeutic effects of LDP may be mediated by up-regulation of CLCF1 gene expression and activation of the JAK/STAT signaling pathway.
Objectives: To investigate the mechanism of Liuwei Dihuang Pill(六味地黄丸, LDP) in treating postmenopausal osteoporosis(PMOP) with Shen(Kidney) yin deficiency. Methods: In this study, 205 cases of PMOP were divided into the PMOP Shen-yin deficiency group(Group A), PMOP Shen-yang deficiency group(Group B), PMOP without Shen deficiency group(Group C), and control group(Group N). Real-time polymerase chain reaction(RT-PCR) and Western blot techniques were used to observe the effects of LDP treatment on the cardiotrophin-like cytokine factor 1(CLCF1), ankyrin repeat and SOCS box containing 1(ASB1), and prokineticin 2(PROK2) genes and the Janus kinase/signal transducer and activator of transcription(JAK/STAT) signaling pathway. Results: The m RNA(P<0.05) and protein(P<0.01) expression levels of the CLCF1 gene in Group A were significantly lower than the corresponding levels in Group N. After LDP treatment for 3 months, the m RNA expression levels of the CLCF1 gene were obviously up-regulated(P<0.01). After 6-month treatment, the expression levels of CLCF1 m RNA and protein were significantly up-regulated(both P<0.01), and the average bone density of the top femur had significantly increased(P<0.05). In vitro, CLCF1 overexpression resulted in a significant increase in the total protein and phosphorylated protein levels of JAK2 and STAT3. Conclusions: The CLCF1 gene is an important gene associated with PMOP Shen-yin deficiency and the therapeutic effects of LDP may be mediated by up-regulation of CLCF1 gene expression and activation of the JAK/STAT signaling pathway.
Objectives: To investigate the mechanism of Liuwei Dihuang Pill(六味地黄丸, LDP) in treating postmenopausal osteoporosis(PMOP) with Shen(Kidney) yin deficiency. Methods: In this study, 205 cases of PMOP were divided into the PMOP Shen-yin deficiency group(Group A), PMOP Shen-yang deficiency group(Group B), PMOP without Shen deficiency group(Group C), and control group(Group N). Real-time polymerase chain reaction(RT-PCR) and Western blot techniques were used to observe the effects of LDP treatment on the cardiotrophin-like cytokine factor 1(CLCF1), ankyrin repeat and SOCS box containing 1(ASB1), and prokineticin 2(PROK2) genes and the Janus kinase/signal transducer and activator of transcription(JAK/STAT) signaling pathway. Results: The m RNA(P<0.05) and protein(P<0.01) expression levels of the CLCF1 gene in Group A were significantly lower than the corresponding levels in Group N. After LDP treatment for 3 months, the m RNA expression levels of the CLCF1 gene were obviously up-regulated(P<0.01). After 6-month treatment, the expression levels of CLCF1 m RNA and protein were significantly up-regulated(both P<0.01), and the average bone density of the top femur had significantly increased(P<0.05). In vitro, CLCF1 overexpression resulted in a significant increase in the total protein and phosphorylated protein levels of JAK2 and STAT3. Conclusions: The CLCF1 gene is an important gene associated with PMOP Shen-yin deficiency and the therapeutic effects of LDP may be mediated by up-regulation of CLCF1 gene expression and activation of the JAK/STAT signaling pathway.
引文
1.Scott LJ.Denosumab:a review of its use in postmenopausal women with osteoporosis.Drugs Aging 2014;31:555-576.
2.Li DT,Li FY,Wang J,Liu JH,Yan N,Cheng YM,etal.Astudy of diagnostic criteria for traditional Chinese medicine syndromes in osteoporosis.J Chin Integr Med(Chin)2011;9:1326-1332.
3.Xia BJ,Tong PJ,Sun Y,Zhou LY,Jin HT.Methods and evaluations on the sterioid-induced osteoporosis mice model with the type of Kidney-yin deficiency.China JOrthop Traumatol(Chin)2014;27:673-679.
4.Xie LH,Zhao YP,Chen K,etal.Bioinformatics analysis of genes expression profiles of postmenopausal osteoporosis with Kidney yin deficiency.J Clin Rehab Tissue Engin Res(Chin)2011;15:2833-2837.
5.Tarca AL,Bhatti G,Romero R.A comparison of gene set analysis methods in terms of sensitivity,prioritization and specificity.PLo S One 2013;8:e79217.
6.Chamorro CI,Zeiai S,Engberg GR,Brodin D,Nordenskj?ld A,Fossum M.A study on proliferation and gene expression in normal human urothelial cells in culture.Tissue Eng Patr A 2015;21:510-517.
7.Xie YM,Yuwen Y,Dong FH,Sun SC,Wang HM,Liu QS,etal.Clinical practice guideline of traditional medicine for primary osteoporosis.Chin J Integr Med 2011;17:52-63.
8.Jiang M,Lu C,Zhang C,Yang J,Tan Y,Lu A,etal.Syndrome differentiation in modern research of traditional Chinese medicine.J Ethnopharmacol 2012;140:634-642.
9.Peacock M,Turner CH,Econs MJ,Foroud T.Genetics of osteoporosis.Endoc Rev 2002;23:303-326.
10.Sugiya N,Nakashima A,Takasugi N,Kawai A,Kiribayashi K,Tanaka J,etal,Endogenous estrogen may prevent bone loss in postmenopausal hemodialysis patients throughout life.Osteoporos Int 2011;22:1573-1579.
11.Nakashima T,Takayanagi H.Osteoimmunology:crosstalk between the immune and bone systems.J Clin Immunol2009;29:555-567.
12.Liu ZH,ed.Osteoporosis research and prevention.Beijing:Chemical Industry Press;1994:226-261.
13.Xia B,Xu B,Sun Y,Xiao L,Pan J,Jin H,etal.The effects of Liuwei Dihuang on canonical Wnt/β-catenin signaling pathway in osteoporosis.J Ethnopharmacol2014;153:133-141.
14.Zhou P,Zhou Q.Observation of the Liuwei Dihuang Pill treatment of 30 cases of osteoporosis in older men.Shanxi J Tradit Chin Med(Chin)2014;35:1191-1192.
15.Zhang HB,Li CY.Effect of Liuwei Dihuang Pill on bone mineral density of the primary osteoporosis(Shen yin deficiency).Chin J Tradit Med Traumatol Orthop(Chin)2011;19:18-20.
16.Fu B,Lun X,Gong Y.Effects of the combined therapy of acupuncture with herbal drugs on male immune infertility-a clinical report of 50 cases.J Tradit Chin Med2005;25:186-189.
17.Erices A,Conget P,Rojas C,Minguell JJ.GP130 activation by soluble interleukin-6 receptor/interleukin-6 enhances osteoblastic differentiation of human bone marrow-derived mesenchymas stem cells.Exp Cell Res 2002;280:24-32.
18.Mihara M,Hashizume M,Yoshida H,Suzuki M,Shiina M.IL-6/IL-6 receptor system and its role in physiological and pathological conditions.Clin Sci 2012;122:143-159.
19.Fang J,Zhang YX,Ru XB,Wei XL.Effect of liuwei dihuang decoction,on the cytokine expression in splenocytes in AArats.China J Chin Mater Med(Chin)2001;26:128-131.
20.Gao D,Zheng LP,Lin JM,Lin W,Chen XZ,Song J,etal.Experimental study on effect of Liuwei Dihuang Pill on HSC from mouse marrow.J Chin Med Mater(Chin)2008;31:251-254.
21.Elsaeidi F,Bemben MA,Zhao XF,Goldman D.Jak/Stat signaling stimulates zebrafish optic nerve regeneration and overcomes the inhibitory actions of Socs3 and Sfpq.JNeurosci 2014;34:2632-2644.
22.Senaldi G,Varnum BC,Sarmiento U,Starnes C,Lile J,Scully S,etal.Novel neurotrophin-1/B cell-stimulating factor-3:a cytokine of the IL-6 family.Proc Natl Acad Sci US A 1999;96:11458-11463.
23.Burger R,Bakker F,Guenther A,Baum W,Schmidt-Arras D,Hideshima T,etal.Functional significance of novel neurotrophin-1/B cell-stimulating factor-3(cardiotrophin-like cytokine)for human myeloma cell growth and survival.Br JHaematol 2003;123:869-878.
24.Elson GC,Lelièvre E,Guillet C,Chevalier S,PlunFavreau H,Froger J,etal.CLF associates with CLC to form a functional heteromeric ligand for the CNTF receptor complex.Nat Neurosci 2000;3:867-872.
25.Moortgat S,Benoit V,Deprez M,Charon A,Maystadt I.A new Turkish infant with clinical features of CS/CISS1syndrome and homozygous CRLF1 mutation.Eur J Med Genet 2014;57:212-215.
26.Hahn AF,Waaler PE,Kvistad PH,Bamforth JS,Miles JH,Mc Leod JG,etal.Cold-induced sweating syndrome CISS1and CISS2:manifestations from infancy to adulthood.Four new cases.J Neurol Sci 2010;293:68-75.
27.Aljabari S,Howard E,Bell T,Vasylyeva TL.Cold induced s w e a t i n g s y n d r o m e w i t h u r i n a r y s y s t e m a n o m a l y association.Case Rep Pediatr 2013;2013:173890.
28.Coskun M,Salem M,Pedersen J,Nielsen OH.Involvement of JAK/STAT signaling in the pathogenesis of inflammatory bowel disease.Pharmacol Res 2013;76:1-8.
29.Joung YH,Lim EJ,Darvin P,Chung SC,Jang JW,Do Park K,etal.MSM enhances GH signaling via the Jak2/STAT5b pathway in osteoblast-like cells and osteoblast differentiation through the activation of STAT5b in MSCs.PLo S One 2012;7:e47477.
30.Huang E,Zhu G,Jiang W,Yang K,Gao Y,Luo Q,etal.Growth hormone synergizes with BMP9 in osteogenic differentiation by activating the JAK/STAT/IGF1 pathway i n m u r i n e m u l t i l i n e a g e c e l l s.J B o n e M i n e r a l R e s2012;27:1566-1575.
31.Li CH,Zhao JX,Sun L,Yao ZQ,Deng XL,Liu R,etal.AG490 inhibits NFATc1 expression and STAT3 activation during RANKL induced osteoclastogenesis.Biochem Biophys Res Commun 2013;435:533-539.
32.Bellido T,Borba VZ,Roberson P,Manolagas SC.Activation of the Janus kinase/STAT(signal transducer and activator of transcription)signal transduction pathway by interleukin-6-type cytokines promotes osteoblast differentiation.Endocrinology 1997;138:3666-3676.
33.Nishimura R,Moriyama K,Yasukawa K,Mundy GR,Yoneda T.Combination of interleukin-6 and soluble interleukin-6 receptors induces differentiation and activation of JAK-STAT and MAP kinase pathways in MG-63 human osteoblastic cells.J Bone Miner Res 1998;13:777-785.
34.Tawara K,Oxford JT,Jorcyk CL.Clinical significance of interleukin(IL)-6 in cancer metastasis to bone:potentialofantiIL-6 therapies.Cancer Manag Res 2011;3:177-189.
35.Itoh S,Udagawa N,Takahashi N,Yoshitake F,Narita H,Ebisu S,etal.A critical role for interleukin-6 familymediated Stat3 activation in osteoblast differentiation and bone formation.Bone 2006;39:505-512.
36.Heinrich PC,Behrmann I,Haan S,Hermanns HM,MüllerNewen G,Schaper F.Principles of interleukin(IL)-6-type cytokine signalling and its regulation.Biochem J2003;374:1-20.
37.Abraham AK,Ramanathan M,Weinstock-Guttman B,Mager DE.Mechanisms of interferon-beta effects on bone homeostasis.Biochem Pharmacol 2009;77:1757-1762.
38.Sun GJ,Guo T,Chen Y,Xu B,Guo JH,Zhao JN.Significant pathways detection in osteoporosis based on the bibliometric network.Eur Rev Med Pharmacol Sci 2013;17:1-7.
39.Platanias CL,Fish EN.Signaling pathways activated by interferons.Exp Hematol 1999;27:1583-1592.
40.González-Navajas J M,Lee J,David M,Raz E.Immunomodulatory functions of typeⅠinterferons.Nat Rev Immunol 2012;12:125-135.
41.Laroche M,Livideanu C,Paul C,Cantagrel A.Interferon alpha and pamidronate in osteoporosis with fracture secondary to mastocytosis.Am J Med 2011;124:776-778.
42.Kim S,Takayanagi H.The regulation of osteoclastogenesis by IFN.Nihon Rinsho 2005;63:1553-1561.
43.de Bruin AM,Voermans C,Nolte MA.Impact of interferon-γon hematopoiesis.Blood 2014;124:2479-2486.
2.Li DT,Li FY,Wang J,Liu JH,Yan N,Cheng YM,etal.Astudy of diagnostic criteria for traditional Chinese medicine syndromes in osteoporosis.J Chin Integr Med(Chin)2011;9:1326-1332.
3.Xia BJ,Tong PJ,Sun Y,Zhou LY,Jin HT.Methods and evaluations on the sterioid-induced osteoporosis mice model with the type of Kidney-yin deficiency.China JOrthop Traumatol(Chin)2014;27:673-679.
4.Xie LH,Zhao YP,Chen K,etal.Bioinformatics analysis of genes expression profiles of postmenopausal osteoporosis with Kidney yin deficiency.J Clin Rehab Tissue Engin Res(Chin)2011;15:2833-2837.
5.Tarca AL,Bhatti G,Romero R.A comparison of gene set analysis methods in terms of sensitivity,prioritization and specificity.PLo S One 2013;8:e79217.
6.Chamorro CI,Zeiai S,Engberg GR,Brodin D,Nordenskj?ld A,Fossum M.A study on proliferation and gene expression in normal human urothelial cells in culture.Tissue Eng Patr A 2015;21:510-517.
7.Xie YM,Yuwen Y,Dong FH,Sun SC,Wang HM,Liu QS,etal.Clinical practice guideline of traditional medicine for primary osteoporosis.Chin J Integr Med 2011;17:52-63.
8.Jiang M,Lu C,Zhang C,Yang J,Tan Y,Lu A,etal.Syndrome differentiation in modern research of traditional Chinese medicine.J Ethnopharmacol 2012;140:634-642.
9.Peacock M,Turner CH,Econs MJ,Foroud T.Genetics of osteoporosis.Endoc Rev 2002;23:303-326.
10.Sugiya N,Nakashima A,Takasugi N,Kawai A,Kiribayashi K,Tanaka J,etal,Endogenous estrogen may prevent bone loss in postmenopausal hemodialysis patients throughout life.Osteoporos Int 2011;22:1573-1579.
11.Nakashima T,Takayanagi H.Osteoimmunology:crosstalk between the immune and bone systems.J Clin Immunol2009;29:555-567.
12.Liu ZH,ed.Osteoporosis research and prevention.Beijing:Chemical Industry Press;1994:226-261.
13.Xia B,Xu B,Sun Y,Xiao L,Pan J,Jin H,etal.The effects of Liuwei Dihuang on canonical Wnt/β-catenin signaling pathway in osteoporosis.J Ethnopharmacol2014;153:133-141.
14.Zhou P,Zhou Q.Observation of the Liuwei Dihuang Pill treatment of 30 cases of osteoporosis in older men.Shanxi J Tradit Chin Med(Chin)2014;35:1191-1192.
15.Zhang HB,Li CY.Effect of Liuwei Dihuang Pill on bone mineral density of the primary osteoporosis(Shen yin deficiency).Chin J Tradit Med Traumatol Orthop(Chin)2011;19:18-20.
16.Fu B,Lun X,Gong Y.Effects of the combined therapy of acupuncture with herbal drugs on male immune infertility-a clinical report of 50 cases.J Tradit Chin Med2005;25:186-189.
17.Erices A,Conget P,Rojas C,Minguell JJ.GP130 activation by soluble interleukin-6 receptor/interleukin-6 enhances osteoblastic differentiation of human bone marrow-derived mesenchymas stem cells.Exp Cell Res 2002;280:24-32.
18.Mihara M,Hashizume M,Yoshida H,Suzuki M,Shiina M.IL-6/IL-6 receptor system and its role in physiological and pathological conditions.Clin Sci 2012;122:143-159.
19.Fang J,Zhang YX,Ru XB,Wei XL.Effect of liuwei dihuang decoction,on the cytokine expression in splenocytes in AArats.China J Chin Mater Med(Chin)2001;26:128-131.
20.Gao D,Zheng LP,Lin JM,Lin W,Chen XZ,Song J,etal.Experimental study on effect of Liuwei Dihuang Pill on HSC from mouse marrow.J Chin Med Mater(Chin)2008;31:251-254.
21.Elsaeidi F,Bemben MA,Zhao XF,Goldman D.Jak/Stat signaling stimulates zebrafish optic nerve regeneration and overcomes the inhibitory actions of Socs3 and Sfpq.JNeurosci 2014;34:2632-2644.
22.Senaldi G,Varnum BC,Sarmiento U,Starnes C,Lile J,Scully S,etal.Novel neurotrophin-1/B cell-stimulating factor-3:a cytokine of the IL-6 family.Proc Natl Acad Sci US A 1999;96:11458-11463.
23.Burger R,Bakker F,Guenther A,Baum W,Schmidt-Arras D,Hideshima T,etal.Functional significance of novel neurotrophin-1/B cell-stimulating factor-3(cardiotrophin-like cytokine)for human myeloma cell growth and survival.Br JHaematol 2003;123:869-878.
24.Elson GC,Lelièvre E,Guillet C,Chevalier S,PlunFavreau H,Froger J,etal.CLF associates with CLC to form a functional heteromeric ligand for the CNTF receptor complex.Nat Neurosci 2000;3:867-872.
25.Moortgat S,Benoit V,Deprez M,Charon A,Maystadt I.A new Turkish infant with clinical features of CS/CISS1syndrome and homozygous CRLF1 mutation.Eur J Med Genet 2014;57:212-215.
26.Hahn AF,Waaler PE,Kvistad PH,Bamforth JS,Miles JH,Mc Leod JG,etal.Cold-induced sweating syndrome CISS1and CISS2:manifestations from infancy to adulthood.Four new cases.J Neurol Sci 2010;293:68-75.
27.Aljabari S,Howard E,Bell T,Vasylyeva TL.Cold induced s w e a t i n g s y n d r o m e w i t h u r i n a r y s y s t e m a n o m a l y association.Case Rep Pediatr 2013;2013:173890.
28.Coskun M,Salem M,Pedersen J,Nielsen OH.Involvement of JAK/STAT signaling in the pathogenesis of inflammatory bowel disease.Pharmacol Res 2013;76:1-8.
29.Joung YH,Lim EJ,Darvin P,Chung SC,Jang JW,Do Park K,etal.MSM enhances GH signaling via the Jak2/STAT5b pathway in osteoblast-like cells and osteoblast differentiation through the activation of STAT5b in MSCs.PLo S One 2012;7:e47477.
30.Huang E,Zhu G,Jiang W,Yang K,Gao Y,Luo Q,etal.Growth hormone synergizes with BMP9 in osteogenic differentiation by activating the JAK/STAT/IGF1 pathway i n m u r i n e m u l t i l i n e a g e c e l l s.J B o n e M i n e r a l R e s2012;27:1566-1575.
31.Li CH,Zhao JX,Sun L,Yao ZQ,Deng XL,Liu R,etal.AG490 inhibits NFATc1 expression and STAT3 activation during RANKL induced osteoclastogenesis.Biochem Biophys Res Commun 2013;435:533-539.
32.Bellido T,Borba VZ,Roberson P,Manolagas SC.Activation of the Janus kinase/STAT(signal transducer and activator of transcription)signal transduction pathway by interleukin-6-type cytokines promotes osteoblast differentiation.Endocrinology 1997;138:3666-3676.
33.Nishimura R,Moriyama K,Yasukawa K,Mundy GR,Yoneda T.Combination of interleukin-6 and soluble interleukin-6 receptors induces differentiation and activation of JAK-STAT and MAP kinase pathways in MG-63 human osteoblastic cells.J Bone Miner Res 1998;13:777-785.
34.Tawara K,Oxford JT,Jorcyk CL.Clinical significance of interleukin(IL)-6 in cancer metastasis to bone:potentialofantiIL-6 therapies.Cancer Manag Res 2011;3:177-189.
35.Itoh S,Udagawa N,Takahashi N,Yoshitake F,Narita H,Ebisu S,etal.A critical role for interleukin-6 familymediated Stat3 activation in osteoblast differentiation and bone formation.Bone 2006;39:505-512.
36.Heinrich PC,Behrmann I,Haan S,Hermanns HM,MüllerNewen G,Schaper F.Principles of interleukin(IL)-6-type cytokine signalling and its regulation.Biochem J2003;374:1-20.
37.Abraham AK,Ramanathan M,Weinstock-Guttman B,Mager DE.Mechanisms of interferon-beta effects on bone homeostasis.Biochem Pharmacol 2009;77:1757-1762.
38.Sun GJ,Guo T,Chen Y,Xu B,Guo JH,Zhao JN.Significant pathways detection in osteoporosis based on the bibliometric network.Eur Rev Med Pharmacol Sci 2013;17:1-7.
39.Platanias CL,Fish EN.Signaling pathways activated by interferons.Exp Hematol 1999;27:1583-1592.
40.González-Navajas J M,Lee J,David M,Raz E.Immunomodulatory functions of typeⅠinterferons.Nat Rev Immunol 2012;12:125-135.
41.Laroche M,Livideanu C,Paul C,Cantagrel A.Interferon alpha and pamidronate in osteoporosis with fracture secondary to mastocytosis.Am J Med 2011;124:776-778.
42.Kim S,Takayanagi H.The regulation of osteoclastogenesis by IFN.Nihon Rinsho 2005;63:1553-1561.
43.de Bruin AM,Voermans C,Nolte MA.Impact of interferon-γon hematopoiesis.Blood 2014;124:2479-2486.