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Demethylzeylasteral (T-96) Treatment Ameliorates Mice Lupus Nephritis Accompanied by Inhibiting Activation of NF-κB Pathway.

Hu Q, Yang C, Wang Q, Zeng H, Qin W - PLoS ONE (2015)

Bottom Line: Over the past 30 years, research has demonstrated that Tripterygium wilfordii Hook F (TWHF) possesses potent anti-inflammatory and immunosuppressive activities, and that demethylzeylasteral (T-96), an extract of TWHF, may be one of the responsible compounds.Moreover, T-96 significantly suppressed phosphorylations of cytoplasmic IKK and nuclear p65.Because of these potent properties, T-96 should be considered as a promising therapeutic drug for LN.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, P.R. China.

ABSTRACT

Background: Inflammation plays a vital role in the pathogenesis in lupus nephritis (LN), which is largely attributable to the activation of nuclear factor kappa B (NF-κB) signal pathway. NF-κB up-regulates pro-inflammatory mediators, such as TNF-α, cyclo-oxygenase-2 (COX-2) and ICAM-1, and promotes macrophage infiltration into renal tissue, further inducing the progression of LN. Over the past 30 years, research has demonstrated that Tripterygium wilfordii Hook F (TWHF) possesses potent anti-inflammatory and immunosuppressive activities, and that demethylzeylasteral (T-96), an extract of TWHF, may be one of the responsible compounds. Here, we investigate the pharmacodynamic role and therapeutic mechanism by which T-96 suppresses inflammation and reduces renal pathology in the lupus-prone MRL/lpr mice.

Methods: Forty-eight MRL/lpr mice were equally randomly divided into 6 groups (1.2, 0.6 or 0.3 mg/10 g T-96, 0.022 pills/10 g kang lang chuang san (one of Traditional Chinese herb as positive control), 0.125 mg/10 g prednisone and 0.1 ml/10 g normal saline as the LN disease control group). Also, eight WT C57BL/6 mice were used as normal control. After treatment by gavage with 0.10 ml/10 g/day volumes for 8 weeks, all mice were sacrificed and renal tissues were collected. The amount of 24 h proteinuria and the levels of anti-dsDNA antibody in serum were assessed respectively at weeks 0, 4 and 8. Inflammation, cytokines and NF-κB levels were assessed by histological examinations, immunohistochemical analyses and Western blot analyses.

Results: In comparison with untreated MRL/lpr mice, mice treated with 1.2 and 0.6 mg/10 g of T-96 showed a significant improvement in 24 h proteinuria and the levels of anti-dsDNA antibody in serum. In addition, T-96 reduced the secretion of pro-inflammatory mediators such as TNF-α, COX-2 and ICAM-1, and the infiltration of macrophages in renal tissue. Moreover, T-96 significantly suppressed phosphorylations of cytoplasmic IKK and nuclear p65.

Conclusion: This study suggests that T-96 exhibits reno-protective effects in LN accompanied by inhibiting the activation of NF-κB, reducing the downstream pro-inflammatory mediators and thus restricting macrophage infiltration. Because of these potent properties, T-96 should be considered as a promising therapeutic drug for LN.

No MeSH data available.


Related in: MedlinePlus

T-96 attenuates renal lesions in MRL/lpr mice.(A-B) Kidneys were collected at week 8, and stained with hematoxylin-eosin stain (H&E) (A) and Periodic Acid-Schiff stain (PAS) (B) (10 x 20). (C-D) The scores of renal lesions in H&E sections (C) and the scores of pathological activity index (AI) in PAS sections (D) were semi-quantitatively measured. Data were expressed as mean ± SD. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.
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pone.0133724.g003: T-96 attenuates renal lesions in MRL/lpr mice.(A-B) Kidneys were collected at week 8, and stained with hematoxylin-eosin stain (H&E) (A) and Periodic Acid-Schiff stain (PAS) (B) (10 x 20). (C-D) The scores of renal lesions in H&E sections (C) and the scores of pathological activity index (AI) in PAS sections (D) were semi-quantitatively measured. Data were expressed as mean ± SD. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

Mentions: To assess inflammation in MRL/lpr mice, we analyzed pathological changes in the renal tissues. H&E and PAS stainings revealed histological abnormalities in glomeruli, tubule-interstitium and in vessels. Glomerulonephritis, including mesangial cell proliferation, mesangium expansion, crescent formation, and necrosis of capillary loops as well as tubule-interstitial lesions and vasculitis were readily detected in normal saline-treated MRL/lpr mice, manifesting a typical model of LN (Fig 3A1, 3B1, 3C and 3D; 2.60 ± 0.55 of renal lesions and 10.50 ± 1.29 of pathological AI). In contrast, T-96 treatment with 1.2 mg/10g notably ameliorated the lesions of renal tissue, with less damage to glomeruli, tubule-interstitium and vessels relative to normal saline-treated MRL/lpr mice (Fig 3A2, 3B2, 3C and 3D; scores of renal lesions p < 0.05, scores of pathological AI p < 0.001). Mice treated with 0.6 mg/10g T-96 tended to have mild glomerulonephritis, cast formation and inflammation in interstitium, with lower scores of renal lesions and pathological AI than the normal saline-treated MRL/lpr mice, though there were no significant differences in the scores of renal lesions (Fig 3A3, 3B3, 3C and 3D; AI p < 0.001). However, 0.3 mg/10g T-96 and Kang lang chuang san treatment didn’t significantly remit renal damage compared with the normal saline-treated MRL/lpr mice (Fig 3A4, 3A5, 3B4, 3B5, 3C and 3D; all p > 0.05). As expected, prednisone treatment markedly attenuated renal pathology, with mild glomerular mesangial proliferation, cast and interstitium infiltration (Fig 3A6, 3B6, 3C and 3D; both p < 0.001). The infiltration of inflammatory cells was further demonstrated by immunohistological staining for CD68+ cells. There was a predominant population of CD68+ macrophages infiltrating the interstitium, and also in and around the glomeruli in the kidney of the normal saline-treated MRL/lpr mice (Fig 4A1). Inversely, in 1.2 and 0.6 mg/10g T-96 groups, the expression of CD68 was obviously down regulated as compared with the normal saline-treated MRL/lpr mice (Fig 4A2, 4A3 and 4C; both p < 0.01). Since IL23, TNF-α, COX-2 and ICAM-1 play essential roles in inflammatory responses, we also examined the levels of these pro-inflammatory mediators in the kidneys. As expected, an enhanced expression of IL23 was clearly detected in the tubules and glomeruli in the normal saline-treated MRL/lpr mice (Fig 4B1 and 4D). In contrast, T-96 from 1.2 to 0.3 mg/10g and Kang lang chuang san substantially reduced expression of IL23 in the tubules and glomeruli in a concentration-dependent manner (Fig 4B2–4B5 and 4D; all p < 0.001). Moreover, a faint expression of IL23 was observed in the tubules and glomeruli of prednisone–treated MRL/lpr mice, with a remarkable decline in the mean density of IL23 (Fig 4B6 and 4D; p < 0.001). Furthermore, the effects of 1.2, 0.6 mg/10g T-96 and prednisone treatment were comparable (Fig 4D, both p > 0.05). As indicated in Fig 5A1, 5B1 and 5C1, TNF-α was localized predominantly in the tubules, whereas COX-2, ICAM-1 were expressed mainly in the glomeruli in normal saline-treated MRL/lpr mice. TNF-α was effectively suppressed by T-96 from 1.2 to 0.3 mg/10g in the tubules of MRL/lpr mice as compared with the normal saline-treated MRL/lpr mice (Fig 5A2–5A4 and 5D, all p < 0.05). Furthermore, treatments of 1.2 and 0.6 mg/10g T-96 significantly reduced COX-2 expression in the glomeruli relative to normal saline-treated MRL/lpr mice (Fig 5B2, 5B3 and 5E; 1.2 mg/10g T-96 p < 0.01 and 0.6 mg/10g T-96 p < 0.05). But 0.3 mg/10g T-96 didn’t significantly reduce TNF-a expression (Fig 5B4 and 5E; p > 0.05). In addition, ICAM-1 was significantly inhibited by 1.2 mg/10g T-96 compared with the normal saline-treated MRL/lpr mice (Fig 5C2 and 5F; p < 0.05). But 0.6 and 0.3 mg/10g T-96 didn’t reduce ICAM-1 expression (Fig 5C3, 5C4 and 5E; both p > 0.05). Our results also indicated that kang lang chuang san markedly reduced the production of TNF-α and ICAM-1 in renal tissues, but it did not reduce COX-2 expression (Fig 5A5, 5B5, 5C5 and 5D and 5E; TNF-α p < 0.01, COX-2 p > 0.05, ICAM-1 p < 0.05). When compared with the normal saline-treated MRL/lpr mice, the secretions of TNF-α, COX-2 and ICAM-1 were significantly reduced in renal tissues of prednisone-treated mice (Fig 5A6, 5B6, 5C6 5D and 5E; all p < 0.05). Collectively, these findings indicate that T-96 is an effective therapy to antagonize renal inflammation during the progression of LN.


Demethylzeylasteral (T-96) Treatment Ameliorates Mice Lupus Nephritis Accompanied by Inhibiting Activation of NF-κB Pathway.

Hu Q, Yang C, Wang Q, Zeng H, Qin W - PLoS ONE (2015)

T-96 attenuates renal lesions in MRL/lpr mice.(A-B) Kidneys were collected at week 8, and stained with hematoxylin-eosin stain (H&E) (A) and Periodic Acid-Schiff stain (PAS) (B) (10 x 20). (C-D) The scores of renal lesions in H&E sections (C) and the scores of pathological activity index (AI) in PAS sections (D) were semi-quantitatively measured. Data were expressed as mean ± SD. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4514757&req=5

pone.0133724.g003: T-96 attenuates renal lesions in MRL/lpr mice.(A-B) Kidneys were collected at week 8, and stained with hematoxylin-eosin stain (H&E) (A) and Periodic Acid-Schiff stain (PAS) (B) (10 x 20). (C-D) The scores of renal lesions in H&E sections (C) and the scores of pathological activity index (AI) in PAS sections (D) were semi-quantitatively measured. Data were expressed as mean ± SD. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.
Mentions: To assess inflammation in MRL/lpr mice, we analyzed pathological changes in the renal tissues. H&E and PAS stainings revealed histological abnormalities in glomeruli, tubule-interstitium and in vessels. Glomerulonephritis, including mesangial cell proliferation, mesangium expansion, crescent formation, and necrosis of capillary loops as well as tubule-interstitial lesions and vasculitis were readily detected in normal saline-treated MRL/lpr mice, manifesting a typical model of LN (Fig 3A1, 3B1, 3C and 3D; 2.60 ± 0.55 of renal lesions and 10.50 ± 1.29 of pathological AI). In contrast, T-96 treatment with 1.2 mg/10g notably ameliorated the lesions of renal tissue, with less damage to glomeruli, tubule-interstitium and vessels relative to normal saline-treated MRL/lpr mice (Fig 3A2, 3B2, 3C and 3D; scores of renal lesions p < 0.05, scores of pathological AI p < 0.001). Mice treated with 0.6 mg/10g T-96 tended to have mild glomerulonephritis, cast formation and inflammation in interstitium, with lower scores of renal lesions and pathological AI than the normal saline-treated MRL/lpr mice, though there were no significant differences in the scores of renal lesions (Fig 3A3, 3B3, 3C and 3D; AI p < 0.001). However, 0.3 mg/10g T-96 and Kang lang chuang san treatment didn’t significantly remit renal damage compared with the normal saline-treated MRL/lpr mice (Fig 3A4, 3A5, 3B4, 3B5, 3C and 3D; all p > 0.05). As expected, prednisone treatment markedly attenuated renal pathology, with mild glomerular mesangial proliferation, cast and interstitium infiltration (Fig 3A6, 3B6, 3C and 3D; both p < 0.001). The infiltration of inflammatory cells was further demonstrated by immunohistological staining for CD68+ cells. There was a predominant population of CD68+ macrophages infiltrating the interstitium, and also in and around the glomeruli in the kidney of the normal saline-treated MRL/lpr mice (Fig 4A1). Inversely, in 1.2 and 0.6 mg/10g T-96 groups, the expression of CD68 was obviously down regulated as compared with the normal saline-treated MRL/lpr mice (Fig 4A2, 4A3 and 4C; both p < 0.01). Since IL23, TNF-α, COX-2 and ICAM-1 play essential roles in inflammatory responses, we also examined the levels of these pro-inflammatory mediators in the kidneys. As expected, an enhanced expression of IL23 was clearly detected in the tubules and glomeruli in the normal saline-treated MRL/lpr mice (Fig 4B1 and 4D). In contrast, T-96 from 1.2 to 0.3 mg/10g and Kang lang chuang san substantially reduced expression of IL23 in the tubules and glomeruli in a concentration-dependent manner (Fig 4B2–4B5 and 4D; all p < 0.001). Moreover, a faint expression of IL23 was observed in the tubules and glomeruli of prednisone–treated MRL/lpr mice, with a remarkable decline in the mean density of IL23 (Fig 4B6 and 4D; p < 0.001). Furthermore, the effects of 1.2, 0.6 mg/10g T-96 and prednisone treatment were comparable (Fig 4D, both p > 0.05). As indicated in Fig 5A1, 5B1 and 5C1, TNF-α was localized predominantly in the tubules, whereas COX-2, ICAM-1 were expressed mainly in the glomeruli in normal saline-treated MRL/lpr mice. TNF-α was effectively suppressed by T-96 from 1.2 to 0.3 mg/10g in the tubules of MRL/lpr mice as compared with the normal saline-treated MRL/lpr mice (Fig 5A2–5A4 and 5D, all p < 0.05). Furthermore, treatments of 1.2 and 0.6 mg/10g T-96 significantly reduced COX-2 expression in the glomeruli relative to normal saline-treated MRL/lpr mice (Fig 5B2, 5B3 and 5E; 1.2 mg/10g T-96 p < 0.01 and 0.6 mg/10g T-96 p < 0.05). But 0.3 mg/10g T-96 didn’t significantly reduce TNF-a expression (Fig 5B4 and 5E; p > 0.05). In addition, ICAM-1 was significantly inhibited by 1.2 mg/10g T-96 compared with the normal saline-treated MRL/lpr mice (Fig 5C2 and 5F; p < 0.05). But 0.6 and 0.3 mg/10g T-96 didn’t reduce ICAM-1 expression (Fig 5C3, 5C4 and 5E; both p > 0.05). Our results also indicated that kang lang chuang san markedly reduced the production of TNF-α and ICAM-1 in renal tissues, but it did not reduce COX-2 expression (Fig 5A5, 5B5, 5C5 and 5D and 5E; TNF-α p < 0.01, COX-2 p > 0.05, ICAM-1 p < 0.05). When compared with the normal saline-treated MRL/lpr mice, the secretions of TNF-α, COX-2 and ICAM-1 were significantly reduced in renal tissues of prednisone-treated mice (Fig 5A6, 5B6, 5C6 5D and 5E; all p < 0.05). Collectively, these findings indicate that T-96 is an effective therapy to antagonize renal inflammation during the progression of LN.

Bottom Line: Over the past 30 years, research has demonstrated that Tripterygium wilfordii Hook F (TWHF) possesses potent anti-inflammatory and immunosuppressive activities, and that demethylzeylasteral (T-96), an extract of TWHF, may be one of the responsible compounds.Moreover, T-96 significantly suppressed phosphorylations of cytoplasmic IKK and nuclear p65.Because of these potent properties, T-96 should be considered as a promising therapeutic drug for LN.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, P.R. China.

ABSTRACT

Background: Inflammation plays a vital role in the pathogenesis in lupus nephritis (LN), which is largely attributable to the activation of nuclear factor kappa B (NF-κB) signal pathway. NF-κB up-regulates pro-inflammatory mediators, such as TNF-α, cyclo-oxygenase-2 (COX-2) and ICAM-1, and promotes macrophage infiltration into renal tissue, further inducing the progression of LN. Over the past 30 years, research has demonstrated that Tripterygium wilfordii Hook F (TWHF) possesses potent anti-inflammatory and immunosuppressive activities, and that demethylzeylasteral (T-96), an extract of TWHF, may be one of the responsible compounds. Here, we investigate the pharmacodynamic role and therapeutic mechanism by which T-96 suppresses inflammation and reduces renal pathology in the lupus-prone MRL/lpr mice.

Methods: Forty-eight MRL/lpr mice were equally randomly divided into 6 groups (1.2, 0.6 or 0.3 mg/10 g T-96, 0.022 pills/10 g kang lang chuang san (one of Traditional Chinese herb as positive control), 0.125 mg/10 g prednisone and 0.1 ml/10 g normal saline as the LN disease control group). Also, eight WT C57BL/6 mice were used as normal control. After treatment by gavage with 0.10 ml/10 g/day volumes for 8 weeks, all mice were sacrificed and renal tissues were collected. The amount of 24 h proteinuria and the levels of anti-dsDNA antibody in serum were assessed respectively at weeks 0, 4 and 8. Inflammation, cytokines and NF-κB levels were assessed by histological examinations, immunohistochemical analyses and Western blot analyses.

Results: In comparison with untreated MRL/lpr mice, mice treated with 1.2 and 0.6 mg/10 g of T-96 showed a significant improvement in 24 h proteinuria and the levels of anti-dsDNA antibody in serum. In addition, T-96 reduced the secretion of pro-inflammatory mediators such as TNF-α, COX-2 and ICAM-1, and the infiltration of macrophages in renal tissue. Moreover, T-96 significantly suppressed phosphorylations of cytoplasmic IKK and nuclear p65.

Conclusion: This study suggests that T-96 exhibits reno-protective effects in LN accompanied by inhibiting the activation of NF-κB, reducing the downstream pro-inflammatory mediators and thus restricting macrophage infiltration. Because of these potent properties, T-96 should be considered as a promising therapeutic drug for LN.

No MeSH data available.


Related in: MedlinePlus