Modified Gexiazhuyu decoction (膈下逐瘀汤加减方) alleviates chronic salpingitis via p38 signaling pathway

doi:10.19852/j.cnki.jtcm.2022.02.003

Abstract

Abstract:

OBJECTIVE: To investigate pharmacodynamic effects of modified Gexiazhuyu decoction (MGXZYD) and explore the underlying mechanism in the treatment of chronic salpingitis

METHODS: Chronic salpingitis model rats were firstly constructed and the blood was collected to detect the whole blood viscosity and plasma viscosity. Rat oviduct were collected to evaluate the macroscopic damage and the pathological injury and fibrosis of oviduct by hematoxylin-eosin (HE) and Masson staining. Elisa assay was to detect the production interleukin-1 β (IL-1β) in serum and collagen I (COL-1), matrix metalloprotein 9 (MMP-9), tissue inhibitor of metalloproteinases 1 (TIMP-1) in oviduct tissue. And immunohistochemical staining with MMP-9 and TIMP-1 in oviduct tissue were examined. Western blot was used to detect the expressions of p38 mitogen-activated protein kinases (p38MAPK), phospho-p38MPAK (p-p38MPAK), transforming growth factor-β1 (TGF-β1) in oviduct. The expression of α-smooth muscle actin (α-SMA), p-p38MPAK, in oviduct tissue were detected by immunofluorescence method. The mRNA of p-p38MAPK, α -SMA, COL-1, MMP-9, TIMP-1 was measured by reverse transcription-polymerase chain reaction.

RESULTS: Rats administrated with MGXZYD demonstrated decreased the whole blood viscosity and plasma viscosity. MGXZYD obviously improved the tubal wall thickening, swelling and pelvic adhesion. And HE and Masson staining showed MGXZYD improved the pathological injury and fibrosis of oviduct. The results of MTT assay and flow cytometry indicated that MGXZYD could decreased the NIN-3T3 cells viability and improved the apoptosis. Besides, MGXZYD inhibited the protein and / or mRNA of TGF-β1, IL-1β, COL-1, α-SMA, p-p38MAPK expressions and increased the production of MMP-9/TIMP-1.

CONCLUSION: MGXZYD could prevent the progression of chronic salpingitis by inhibited the fibrocyte and inflammation which via inhibited the p38 MAPK signaling pathway.

Key words: salpingitis, fibrosis, p38 mitogen-activated protein kinases, inflammation, modified Gexiazhuyu decoction

HU Xijiao, LI Shuoxi, YANG Dongxia, GU Na, LIU Jinzhe, WANG Yawen, LIU Li, SUN Yiming. Modified Gexiazhuyu decoction (膈下逐瘀汤加减方) alleviates chronic salpingitis via p38 signaling pathway[J]. Journal of Traditional Chinese Medicine, 2022, 42(2): 213-220.

Figures/Tables 5

Table 1 Macroscopic observation of oviduct (n)

Group	n	Swollen veins
Group	n	-	+	++		-	+	++				-		+		++
Control	20	16	3	1	16		2		2	14			4		2
Model	20	0	6	14	2		6		12		0			4	16
MGXY-H	20	8	6	6	12		4		4		10			4	8
MGXY-M	20	6	8	4	10		8		2		6			8	6
MGXY-L	20	8	6	6	6		6		6		3			8	9

Figure 1 Histopathological examination of oviduct tissue (×100) A-B: oviduct tissue with hematoxylin-eosin staining (A) and Masson staining (B). Control group and model group were given distilled water 2 mL/100 g weight. MGXZY-H, M, L groups given the MGXZY (1.5, 0.75, 0.38 g/mL), respectively, lasted for 4 weeks. MGXZY: modified Gexiazhuyu decoction.

Figure 2 MGXZYD inhibited the production of TGF-β1, IL-1β, COL-1 and α-SMA A, B: expression of TGF-β1 were detected by Western blot (A) and statistical analysis on it (B). C, E: ELISA kit was used to determine the generations of IL-1β and COL-1. D, F: expression of α-SMA, COL-1 mRNA was measured by RT-PCR. G: immunofluorescence was used to measure the expression of α-SMA. Control group and model group were given distilled water 2 mL/100 g weight. MGXZY-H, M, L groups given the MGXZY (1.5, 0.75, 0.38 g/mL), respectively, lasted for 4 weeks. MGXZYD: modified Gexiazhuyu decoction; TGF-β1: transforming growth factor-β1; ELISA: enzyme-linked immunosorbent assay; IL-1β: interleukin-1β; α-SMA: alpha-smooth muscle actin; COL-1: recombinant collagen typeⅠ; RT-PCR: reverse transcription-polymerase chain reaction. Data are expressed as mean ± standard deviation of three independent experiments. aP < 0.01, compared with control group; bP < 0.01, cP < 0.05, compared with model group.

Figure 3 MGXZYD regulated the expression of MMP-9 and TIPM-1 A-C: the productions of MMP-9 and TIMP-1 were detected by ELISA Kit, and statistics on ratio of MMP-9/TIPM-1; D, E: expression of MMP-9 and TIMP-1 mRNA by using RT-PCR; F: expression of MMP-9 and TIMP-1 in oviduct tissues was examined by immunohistochemical analysis ( × 400, brown yellow granules indicate positive reaction). Control group and model group were given distilled water 2 mL/100 g weight. MGXZY-H, M, L groups given the MGXZY (1.5, 0.75, 0.38 g/mL), respectively, lasted for 4 weeks. MGXZYD: modified Gexiazhuyu decoction; MMP: matrix metalloproteinase; TIMP: tissue inhibitor of metalloproteinase; ELISA: enzyme-linked immunosorbent assay; RT-PCR: reverse transcription-polymerase chain reaction. Data are expressed as mean ± standard deviation of three independent experiments. aP < 0.01, compared with control group; bP < 0.01, compared with model group.

Figure 4 MGXZYD inhibited the expression of p-p38 Control group and model group were given distilled water 2 mL / 100 g weight. MGXZY-H, M, L groups given the MGXZY (1.5, 0.75, 0.38 g/mL), respectively, lasted for 4 weeks. A: Western bot was used to detect the expression of p-38 and p-p38; B-D: statistical data on the p38/β-actin, p-p38/β-actin, p-p38/p38; E: p-p38 mRNA was measured by RT-PCR; F: immunohistochemical image of p-p38 measured by immunofluorescence technique. MGXZYD: modified Gexiazhuyu decoction; RT-PCR: reverse transcription-polymerase chain reaction; p38 MAPK: p38 mitogen-activated protein kinase. Data are expressed as mean ± standard deviation of three independent experiments. aP < 0.01, compared with control group; bP < 0.01, cP < 0.05, compared with model group.

References 39.

1.	Dun EC, Nezhat CH. Tubal factor infertility: diagnosis and management in the era of assisted reproductive technology. Obstet Gynecol Clin North Am 2012; 39: 551-66. DOI URL
2.	Barkwill D, Tobler KJ. Salpingitis isthmica nodosa. In: StatPearls. edn. Treasure Island: Statpearls Publishing, 2020: 77-80.
3.	Wiesenfeld HC, Hillier SL, Krohn MA, et al. Lower genital tract infection and endometritis: insight into subclinical pelvic inflammatory disease. Obstet Gynecol 2002; 100: 456-63. PMID
4.	Li C, Meng CX, Sun LL, et al. Reduced prevalence of chronic tubal inflammation in tubal pregnancies after levonorgestrel emergency contraception failure. Pharmacoepidemiol Drug Saf 2015; 24: 548-54. DOI URL
5.	Avery D, Govindaraju P, Jacob M, Todd L, Monslow J, Pure E. Extracellular matrix directs phenotypic heterogeneity of activated fibroblasts. Matrix Biol 2018; 67: 90-106. DOI URL
6.	Humphreys BD, Lin SL, Kobayashi A, et al. Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis. Am J Pathol 2010; 176: 85-97. DOI PMID
7.	Liu Y. Cellular and molecular mechanisms of renal fibrosis. Nat Rev Nephrol 2011; 7: 684-96. DOI URL
8.	Vernon MA, Mylonas KJ, Hughes J. Macrophages and renal fibrosis. Semin Nephrol 2010; 30: 302-17. DOI URL
9.	Grande MT, Perez-Barriocanal F, Lopez-Novoa JM. Role of inflammation in tubulo-interstitial damage associated to obstructive nephropathy. J Inflamm (Lond) 2010; 7: 19. DOI URL
10.	Karsdal MA, Nielsen SH, Leeming DJ, et al. The good and the bad collagens of fibrosis - their role in signaling and organ function. Adv Drug Deliv Rev 2017; 121: 43-56. DOI URL
11.	Terzic M, Kocijancic D. Pelvic inflammatory disease: contemporary diagnostic and therapeutic approach. Srp Arh Celok Lek 2010; 138: 658-63. DOI URL
12.	Liu Q, Zhuang Y, Ouyang N, Yu H. Cytochalasin D promotes osteogenic differentiation of MC3T3-E1 cells via p38-MAPK signaling pathway. Curr Mol Med 2019; 20: 79-88. DOI URL
13.	Ji L, Wang T, Tian L, Song H, Gao M. Roxatidine inhibits fibrosis by inhibiting NF kappa B and MAPK signaling in macrophages sensing breast implant surface materials. Mol Med Rep 2020; 21: 161-72.
14.	Jin YK, Li XH, Wang W, et al. Follistatin-Like 1 promotes bleomycin-induced pulmonary fibrosis through the transforming growth factor beta 1/Mitogen-activated protein kinase signaling pathway. Chin Med J (Engl) 2018; 131: 1917-25.
15.	Sun H, Ding JM, Zheng HH, et al. The effects of Sidt2 on the inflammatory pathway in mouse mesangial cells. Mediators Inflamm 2020; 2020: 3560793.
16.	Chen JY, Chen HL, Cheng JC, et al. A Chinese herbal medicine, Gexia-Zhuyu Tang (GZT), prevents dimethylnitrosamine-induced liver fibrosis through inhibition of hepatic stellate cells proliferation. J Ethnopharmacol 2012; 142: 811-8. DOI URL
17.	Deng Z, Zhang S, Ge S, Kong F, Cao S, Pan Z. Gexia-Zhuyu decoction attenuates carbon tetrachloride-induced liver fibrosis in mice partly via liver angiogenesis mediated by myeloid cells. Med Sci Monit 2019; 25: 2835-44. DOI URL
18.	Li Z, Zhang Z, Chen X, Zhou J, Xiao XM. Treatment evaluation of Wharton's jelly-derived mesenchymal stem cells using a chronic salpingitis model: an animal experiment. Stem Cell Res Ther 2017; 8: 232. DOI URL
19.	Bonfante F, Mazzetto E, Zanardello C, et al. A G1-lineage H9N2 virus with oviduct tropism causes chronic pathological changes in the infundibulum and a long-lasting drop in egg production. Vet Res 2018; 49: 83. DOI URL
20.	Ma K, Wang KL, Chen YX. Infertility caused by salpingitis treated based on theory of kidney deficiency and blood stasis. Zhong Guo Zhong Yao Za Zhi 2019; 44: 1099-103.
21.	Zhao Z, Yu H, Peng Y, et al. Comparison of effect of formulas clearing away heat and promoting blood circulation on prevention and treatment of liver fibrosis in CCl₄ mice. Zhong Guo Zhong Yao Za Zhi 2012; 37: 1804-8.
22.	Hafner LM. Pathogenesis of fallopian tube damage caused by Chlamydia trachomatis infections. Contraception 2015, 92: 108-15. DOI URL
23.	Zhou F, Shi LB, Zhang SY. Ovarian fibrosis: a phenomenon of concern. Chin Med J (Engl) 2017; 130: 365-71.
24.	Manetti M, Romano E, Rosa I, et al. Endothelial-to-mesenchymal transition contributes to endothelial dysfunction and dermal fibrosis in systemic sclerosis. Ann Rheum Dis 2017; 76: 924-34. DOI PMID
25.	Rubis P, Wisniowska-Smialek S, Wypasek E, et al. Fibrosis of extracellular matrix is related to the duration of the disease but is unrelated to the dynamics of collagen metabolism in dilated cardiomyopathy. Inflamm Res 2016; 65: 941-9. DOI URL
26.	Cui N, Hu M, Khalil RA. Biochemical and biological attributes of Matrix Metalloproteinases. Prog Mol Biol Transl Sci 2017; 147: 1-73.
27.	Mitrut R, Stepan AE, Margaritescu C, et al. Immunoexpression of MMP-8, MMP-9 and TIMP-2 in dilated cardiomyopathy. Rom J Morphol Embryol 2019; 60: 119-24.
28.	Arpino V, Brock M, Gill SE, The role of TIMPs in regulation of extracellular matrix proteolysis. Matrix Biol 2015; 44- 6: 247-54.
29.	Juica NE, Rodas PI, Solar P, et al. Neisseria gonorrhoeae challenge increases matrix metalloproteinase-8 expression in fallopian tube explants. Front Cell Infect Microbiol 2017; 7: 399. DOI URL
30.	Wu Y, Deng J, Rychahou PG, Qiu S, Evers BM, Zhou BP. Stabilization of snail by NF-kappa B is required for inflammation-induced cell migration and invasion. Cancer Cell 2009; 15: 416-28. DOI URL
31.	Bristol-Gould SK, Hutten CG, Sturgis C, Kilen SM, Mayo KE, Woodruff TK. The development of a mouse model of ovarian endosalpingiosis. Endocrinology 2005; 146: 5228-36. PMID
32.	Witkin SS, Toth M, Jeremias J, Ledger WJ. Increased inducibility of inflammatory mediators from peripheral blood mononuclear cells of women with salpingitis. Am J Obstet Gynecol 1991; 165: 719-23. PMID
33.	Kim KK, Sheppard D, Chapman HA. TGF-beta1 signaling and tissue fibrosis. Cold Spring Harb Perspect Biol 2018; 10: a022293. DOI URL
34.	Kahari VM, Heino J, Vuorio E. Interleukin-1 increases collagen production and mRNA levels in cultured skin fibroblasts. Biochim Biophys Acta 1987; 929: 142-7.
35.	Kulasekaran P, Scavone CA, Rogers DS, Arenberg DA, Thannickal VJ, Horowitz JC. Endothelin-1 and transforming growth factor-beta1 independently induce fibroblast resistance to apoptosis via AKT activation. Am J Respir Cell Mol Biol 2009; 41: 484-93. DOI URL
36.	Lea S, Harbron C, Khan N, Booth G, Armstrong J, Singh D. Corticosteroid insensitive alveolar macrophages from asthma patients; synergistic interaction with a p38 mitogen-activated protein kinase (MAPK) inhibitor. Br J Clin Pharmacol 2015; 79: 756-66. DOI URL
37.	Yang Y, Kim SC, Yu T, et al. Functional roles of p38 mitogen-activated protein kinase in macrophage-mediated inflammatory responses. Mediators Inflamm 2014; 2014: 352371.
38.	ACOG Committee on Practice Bulletins--Gynecology. ACOG practice bulletin No. 51. Chronic pelvic pain. Obstet Gynecol 2004, 103: 589-605. DOI URL
39.	Disel U, Paydas S, Dogan A, Gulfiliz G, Yavuz S. Effect of colchicine on cyclosporine nephrotoxicity, reduction of TGF-beta overexpression, apoptosis, and oxidative damage: an experimental animal study. Transplant Proc 2004; 36: 1372-6. DOI URL

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