Journal of Traditional Chinese Medicine ›› 2023, Vol. 43 ›› Issue (2): 265-273.DOI: 10.19852/j.cnki.jtcm.20230201.002
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Gouqizi (Fructus Lycii) seed oil reduces D-galactose induced inflammation in testis of rats via Janus kinase 1/signal transducerand activator of transcription 1/nuclear factor-κB in vitro and in vivo
YANG Zhangjie1, WANG Yuxin2, CHEN Dongmei3, ZHAO Shuai4, HU Na4, MA Lianghong5(
), MA Huiming4()
- 1 Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education in Ningxia Medical University, Yinchuan 750004, China; Department of urology, Shenzhen people’s hospital, Shenzhen 518000, China
2 Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education in Ningxia Medical University, Yinchuan 750004, China; Hubei Provincial Hospital of Integrated Chinese and Western Medicine, Hubei 430000, China
3 Ningxia Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Human Stem Cell Research, the General Hospital of Ningxia Medical University, Yinchuan 750004, China
4 Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education in Ningxia Medical University, Yinchuan 750004, China
5 Department of Urology, the General Hospital of Ningxia Medical University, Yinchuan 750004, China
-
Received:2022-01-09Accepted:2022-04-10Online:2023-04-15Published:2023-03-14 -
Contact:MA Huiming, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education in Ningxia Medical University, Yinchuan 750004, China. mhm289@nxmu.edu.cn; MA Lianghong, Department of Urology, the General Hospital of Ningxia Medical University, Yinchuan 750000, China. mmm1770@163.com. Telephone: +86-15226278505 -
Supported by:Natural Science Foundation-funded Project: Activation of SIRT3 by Gouqizi (Fructus Lycii) Seed Oil to Improve Mitochondrial Function and Maintain Homeostasis in Injured Testis of Sub-acutely Aging Male Rats(81860876);Ningxia Natural Science Foundation-funded Project: Effects of Active Components of Gouqizi (Fructus Lycii) Seed Oil on Regulating Autophagy and Inflammation and Delaying Testicular Aging in Sub-acutely Aging Male Rats(2020AAC02016)
Cite this article
YANG Zhangjie, WANG Yuxin, CHEN Dongmei, ZHAO Shuai, HU Na, MA Lianghong, MA Huiming. Gouqizi (Fructus Lycii) seed oil reduces D-galactose induced inflammation in testis of rats via Janus kinase 1/signal transducerand activator of transcription 1/nuclear factor-κB in vitro and in vivo[J]. Journal of Traditional Chinese Medicine, 2023, 43(2): 265-273.
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Figure 1 Effect of FLSO on anti-inflammation in TM4 cells A: number of TM4 cells was measured by CCK-8; B: immunoblot assay for determining inflammatory factors; C: quantification of IL-1β; D: quantification of IL-6; E: quantification of TNF-α; F: quantification of HO-1; G: quantification of IL-10. Densitometry was used to compare the expression levels. β-actin was used as an internal loading control. FLSO: Fructus Lycii seed oil; CCK-8: cell counting kit-8; IL-1β: interleukin 1 beta; IL-6: interleukin 6; TNF-α: tumor necrosis factor α; HO-1: heme oxygenase-1; IL-10: interleukin 10. FLSO groups: 50, 100 and 150 μg/mL FLSO pre-treated for 6 h, and then 200 mmol/L D-galactose was added for 48 h. Data are expressed as the mean ± standard deviaition, n = 3, aP < 0.001, bP < 0.05, cP < 0.01, as compared to the aging model without FLSO.
Figure 1 Effect of FLSO on anti-inflammation in TM4 cells A: number of TM4 cells was measured by CCK-8; B: immunoblot assay for determining inflammatory factors; C: quantification of IL-1β; D: quantification of IL-6; E: quantification of TNF-α; F: quantification of HO-1; G: quantification of IL-10. Densitometry was used to compare the expression levels. β-actin was used as an internal loading control. FLSO: Fructus Lycii seed oil; CCK-8: cell counting kit-8; IL-1β: interleukin 1 beta; IL-6: interleukin 6; TNF-α: tumor necrosis factor α; HO-1: heme oxygenase-1; IL-10: interleukin 10. FLSO groups: 50, 100 and 150 μg/mL FLSO pre-treated for 6 h, and then 200 mmol/L D-galactose was added for 48 h. Data are expressed as the mean ± standard deviaition, n = 3, aP < 0.001, bP < 0.05, cP < 0.01, as compared to the aging model without FLSO.
Figure 2 Expression of NF-κb p65 were inhibited in TM4 cells of FLSO treatment A: expression of NF-κb p65(green) was detected by immunofluorescence in control group (A1, A2, A3); aging model group (A4, A5, A6); FLSO group (A7, A8, A9) (×200); B: immunoblot assay for determining NF-κB p65 phosphorylation; C: quantification of p-P65/P65; D: quantification of p-P65/β-actin. The nucleus of cells with blue color stained by DAPI. Densitometry was used to compare the expression levels. NF-κB: nuclear factor kappa-B; DAPI: 4',6-diamidino-2-phenylindole. Control: normal culture; aging model: 200 mmol/L D-galactose was added for 48 h; FLSO: 100 μg/mL FLSO for 6 h. Data are expressed as the mean ± standard deviaition, n = 3; aP < 0.05, bP < 0.01 as compared to the aging model.
Figure 2 Expression of NF-κb p65 were inhibited in TM4 cells of FLSO treatment A: expression of NF-κb p65(green) was detected by immunofluorescence in control group (A1, A2, A3); aging model group (A4, A5, A6); FLSO group (A7, A8, A9) (×200); B: immunoblot assay for determining NF-κB p65 phosphorylation; C: quantification of p-P65/P65; D: quantification of p-P65/β-actin. The nucleus of cells with blue color stained by DAPI. Densitometry was used to compare the expression levels. NF-κB: nuclear factor kappa-B; DAPI: 4',6-diamidino-2-phenylindole. Control: normal culture; aging model: 200 mmol/L D-galactose was added for 48 h; FLSO: 100 μg/mL FLSO for 6 h. Data are expressed as the mean ± standard deviaition, n = 3; aP < 0.05, bP < 0.01 as compared to the aging model.
Table 1 Changes in inflammatory factors count in rats serum (pg/mL, $\bar{x}\pm s$)
| Item | Control (n = 10) | High dose FLSO (n = 10) | Medium dose FLSO (n = 10) | Low dose FLSO (n = 10) | Aging model (n = 10) |
|---|---|---|---|---|---|
| IL-1β | 61±14a | 66±9a | 61±15a | 89±13 | 99±14 |
| IL-6 | 296±103b | 298±84 | 312±80 | 393±81 | 411±96 |
| IL-10 | 100±15b | 93±12 | 97±13b | 82±16 | 75±15 |
| TNF-α | 56±12b | 54±17a | 60±13b | 76±15 | 81±15 |
Table 1 Changes in inflammatory factors count in rats serum (pg/mL, $\bar{x}\pm s$)
| Item | Control (n = 10) | High dose FLSO (n = 10) | Medium dose FLSO (n = 10) | Low dose FLSO (n = 10) | Aging model (n = 10) |
|---|---|---|---|---|---|
| IL-1β | 61±14a | 66±9a | 61±15a | 89±13 | 99±14 |
| IL-6 | 296±103b | 298±84 | 312±80 | 393±81 | 411±96 |
| IL-10 | 100±15b | 93±12 | 97±13b | 82±16 | 75±15 |
| TNF-α | 56±12b | 54±17a | 60±13b | 76±15 | 81±15 |
Figure 3 Function of testis were protected by FLSO via inflammatory pathways A: histomorphological changes in the testicular tissue of rats treated with FLSO (HE stain, × 200). A1: normal control; A2: aging model group; A3: FLSO group; B: grades of testicular tissues depend on the Johnsen score; C: the endocrine testosterone in the serum of rats was detected; D: the secreted cytokine level of INHB in the serum of rats was measured; E: immunoblot assay for determining JAK1/STAT1 pathway; F: the relative expression of JAK-1; G: the relative expression of p-JAK-1; H: the relative expression of STAT1; I: the relative expression of p-STAT1. Densitometry was used to compare the expression levels. β-actin was used as an internal loading control. INHB: inhibin B; JAK-1: Janus kinase 1; STAT1: signal transducer and activator of transcription 1. Aging model: subcutaneous injection of D-gal at 125 mg·kg--1·d-1 for 8 weeks; control group: equivalent volume of normal saline; FLSO group: intragastric administration of FLSO at 1 mL·kg--1·d-1. Data are expressed as the mean ± standard deviaition, n = 3; aP < 0.01, bP < 0.05 as compared to the aging model.
Figure 3 Function of testis were protected by FLSO via inflammatory pathways A: histomorphological changes in the testicular tissue of rats treated with FLSO (HE stain, × 200). A1: normal control; A2: aging model group; A3: FLSO group; B: grades of testicular tissues depend on the Johnsen score; C: the endocrine testosterone in the serum of rats was detected; D: the secreted cytokine level of INHB in the serum of rats was measured; E: immunoblot assay for determining JAK1/STAT1 pathway; F: the relative expression of JAK-1; G: the relative expression of p-JAK-1; H: the relative expression of STAT1; I: the relative expression of p-STAT1. Densitometry was used to compare the expression levels. β-actin was used as an internal loading control. INHB: inhibin B; JAK-1: Janus kinase 1; STAT1: signal transducer and activator of transcription 1. Aging model: subcutaneous injection of D-gal at 125 mg·kg--1·d-1 for 8 weeks; control group: equivalent volume of normal saline; FLSO group: intragastric administration of FLSO at 1 mL·kg--1·d-1. Data are expressed as the mean ± standard deviaition, n = 3; aP < 0.01, bP < 0.05 as compared to the aging model.
Figure 4 FLSO inhibited the expression of NF-κB p65 in vivo A: the location of Sertoli cells was labeled by specific marker WT1(red), and NF-κB p65 (green) was localized in the testicular tissue by immunofluorescence in control group (A1, A4, A7, A10); aging model group (A2, A5, A8, A11); FLSO group (A3, A6, A9, A12) (×200); B: immunoblot assay for determining NF-κb p65 phosphorylation; C: expression of p-P65/P65 was quantified; D: expression of p-P65/β-actin was quantified. The nucleus of cells with blue color stained by DAPI. Densitometry was used to compare the expression levels. β-actin was used as an internal loading control. WT1: wilm tumor gene1; NF-κB: nuclear factor kappa-B. Aging model: subcutaneous injection of D-gal at 125 mg·kg-1·d-1 for 8 weeks; control group: equivalent volume of normal saline; FLSO group: intragastric administration of FLSO at 1 mL·kg-1·d-1. Data are expressed as the mean ± standard deviaition, n = 3; aP < 0.05, bP < 0.01 as compared to the aging model.
Figure 4 FLSO inhibited the expression of NF-κB p65 in vivo A: the location of Sertoli cells was labeled by specific marker WT1(red), and NF-κB p65 (green) was localized in the testicular tissue by immunofluorescence in control group (A1, A4, A7, A10); aging model group (A2, A5, A8, A11); FLSO group (A3, A6, A9, A12) (×200); B: immunoblot assay for determining NF-κb p65 phosphorylation; C: expression of p-P65/P65 was quantified; D: expression of p-P65/β-actin was quantified. The nucleus of cells with blue color stained by DAPI. Densitometry was used to compare the expression levels. β-actin was used as an internal loading control. WT1: wilm tumor gene1; NF-κB: nuclear factor kappa-B. Aging model: subcutaneous injection of D-gal at 125 mg·kg-1·d-1 for 8 weeks; control group: equivalent volume of normal saline; FLSO group: intragastric administration of FLSO at 1 mL·kg-1·d-1. Data are expressed as the mean ± standard deviaition, n = 3; aP < 0.05, bP < 0.01 as compared to the aging model.
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