Micheliolide ameliorates renal fibrosis by suppressing the Mtdh/BMP/MAPK pathway

Abstract

Micheliolide (MCL), derived from parthenolide (PTL), is known for its antioxidant and anti-inflammatory effects and has multiple roles in inflammatory diseases and tumours. To investigate its effect on renal disease, we intragastrically administrated DMAMCL, a dimethylamino Michael adduct of MCL for in vivo use, in two renal fibrosis models-the unilateral ureteral occlusion (UUO) model and an ischaemia-reperfusion injury (IRI) model and used MCL in combination with transforming growth factor beta 1 (TGF-β1) on mouse tubular epithelial cells (mTEC) in vitro. The expression of fibrotic markers (fibronectin and α-SMA) was remarkably reduced, while the expression of the epithelial marker E-cadherin was restored after DMAMCL treatment both in the UUO and IRI mice. MCL function in TGF-β1-induced epithelial-mesenchymal transition (EMT) in mTEC was consistent with the in vivo results. Metadherin (Mtdh) was activated in the fibrotic condition, suggesting that it might be involved in fibrogenesis. Interestingly, we found that while Mtdh was upregulated in the fibrotic condition, DMAMCL/MCL could suppress its expression. The overexpression of Mtdh exerted a pro-fibrotic effect by modulating the BMP/MAPK pathway in mTECs, and MCL could specifically reverse this effect. In conclusion, DMAMCL/MCL treatment represents a novel and effective therapy for renal fibrosis by suppressing the Mtdh/BMP/MAPK pathway.

Fig. 1

Chemical structures and cytotoxicity of DMAMCL/MCL. a Chemical structures of MCL and DMAMCL. b Evaluation of the cytotoxic effects of MCL on the viability of mTECs. mTECs were incubated with different concentrations of MCL for 24 h, and cell viability was determined using an MTT assay. The data are presented as the mean ± SEM of at least three independent experiments

Fig. 2

DMAMCL attenuates renal fibrosis in UUO mice. a Representative micrographs of Masson’s trichrome staining and IHC staining for α-SMA, fibronectin, and E-cadherin in the obstructed kidneys. Scale bar in Masson’s trichrome staining is 100 μm; in IHC staining is 50 μm. b Representative bands from Western blot analyses of the levels of the α-SMA, fibronectin, and E-cadherin proteins in kidney tissues from the UUO mice. c Relative protein levels as determined by the Western blot assay (b). *P < 0.05 compared with the sham group; #P < 0.05 compared with the vehicle group; ##P < 0.01 compared with the vehicle group (n = 6 mice per group). d Quantification of renal tubular interstitial fibrotic score. ****P < 0.0001 compared with the sham group; ###P < 0.001 compared with the vehicle group (n = 6 mice per group). The data are presented as the mean ± SEM of at least three independent experiments

Fig. 3

DMAMCL protects kidney from fibrosis in the IRI mice. a Representative micrographs of Masson’s trichrome staining and IHC staining for α-SMA, fibronectin, and E-cadherin in the injured kidneys. Scale bar in Masson’s trichrome staining is 100 μm; in IHC staining is 50 μm. b Quantification of renal tubular interstitial fibrotic score. ****P < 0.0001 compared with the sham group; #P < 0.05 compared with the vehicle group. c Serum creatine level in the IRI mice. ****P < 0.0001 compared with the sham group; ##P < 0.01 compared with the vehicle group. d Serum urea level in the IRI mice. ****P < 0.0001 compared with the sham group; ###P < 0.001 compared with the vehicle group. e Representative bands from Western blot analyses of the levels of the α-SMA, fibronectin, and E-cadherin proteins in kidney tissues from the IRI mice. f Relative protein levels as determined by the Western blot assay (e). ***P < 0.001 compared with the sham group; ##P < 0.01 compared with the vehicle group. n = 6 mice per group, all the data are presented as the mean ± SEM of at least three independent experiments

Fig. 4

DMAMCL/MCL Inhibited Mtdh Expression in the UUO and IRI mice. a Representative Western blot showing Mtdh levels in the UUO mice. b Relative levels of the Mtdh protein in the blot shown in (a). **P < 0.01 compared with the sham group; #P < 0.05 compared with the vehicle group. c Representative Western blot showing Mtdh levels in the IRI mice. d Relative levels of the Mtdh protein in the blot shown in (c). ***P < 0.001 compared with the sham group; ###P < 0.001 compared with the vehicle group. e Representative Mtdh IHC staining in the UUO mice. f Representative Mtdh IHC staining in the IRI mice. Scale bar in (e) and (f) represents 50 μm. All the data are presented as the mean ± SEM of at least three independent experiments

Fig. 5

DMAMCL/MCL Inhibited both Mtdh and EMT in vitro. a Representative Western blot shows Mtdh and EMT markers (fibronectin, α-SMA, and E-cadherin) in mTECs cell lysates treated with TGF-β1 (5 ng/ml) for 0, 12, 24, 48 h. b Relative levels of the Mtdh and EMT marker proteins compared to GAPDH. *P < 0.05 compared with 0 h; **P < 0.01 compared with 0 h. c mTECs were coincubated with MCL (0, 1.25, 2.5, 5, or 10 μM) and TGF-β1 (5 ng/ml). Cells were harvested 48 h later, and cell lysates were immunoblotted to detect Mtdh expression. d Relative levels of the Mtdh, proteins compared to GAPDH. *P < 0.05 compared with the control group; #P < 0.05 compared with TGF-β1 group. e Representative Western blot on EMT markers in MCL- TGF-β1 coincubation cell lysates samples. f Relative levels of the EMT markers in the blot shown in (e). *P < 0.05 compared with the control group; #P < 0.05 compared with the TGF-β1 group; ####P < 0.0001 compared with the TGF-β1 group. The data are presented as the mean ± SEM of at least three independent experiments

Fig. 6

Mtdh Promotes EMT in mTECs. a Representative Western blot showing Mtdh levels in LV-Mtdh and LV-NC cells. b Relative levels of Mtdh in these two groups. *P < 0.05 compared with LV-NC. c Representative Western blots showing levels of EMT markers in LV-Mtdh and LV-NC cells. d Relative protein expression shown in (c). *P < 0.05 compared with LV-NC; **P < 0.01 compared with LV-NC; ***P < 0.001 compared with LV-NC. The data are presented as the mean ± SEM of at least three independent experiments

Fig. 7

Knockdown of Mtdh Relieved the TGF-β1-induced EMT in mTECs. a Representative Western blot analyses of sh-NC and sh-Mtdh cells. b, c, d, e and f Relative protein expression shown in (a). *P < 0.05 compared with sh-NC; #P < 0.05 compared with sh-NC + TGF-β1; ##P < 0.01 compared with sh-NC + TGF-β1. The data are presented as the mean ± SEM of at least three independent experiments

Fig. 8

MCL Relieves the Fibrotic Phenotype Induced by Mtdh Overexpression. a Representative Western blot analyses of levels of Mtdh and EMT markers. b, c, d, e and f Relative protein expression shown in (a). *P < 0.05 compared with the NC group; **P < 0.01 compared with the NC group; #P < 0.05 compared with the Mtdh vector group; ##P < 0.01 compared with the Mtdh vector group; ###P < 0.001 compared with the Mtdh vector group; #### P < 0.0001 compared with the Mtdh vector group. The data are presented as the mean ± SEM of at least three independent experiments

Fig. 9

Mtdh Activates BMP/MAPK Signalling. a Representative Western blot analyses of phosphorylated P38 MAPK. *P < 0.05 compared with LV-NC. b Representative Western blot analyses of phosphorylated ERK. **P < 0.01 compared withLV-NC. c Representative Western blot analyses of phosphorylated Smad1/5/9. **P < 0.01 compared with LV-NC. d Representative Western blot analyses of phosphorylated Smad2/3. ns indicates P > 0.05 compared with LV-NC. e Representative Western blot analyses of BMPR1A. **P < 0.01 compared with LV-NC. The data are presented as the mean ± SEM of at least three independent experiments

Fig. 10

Mtdh knockdown inhibits TGF-β1-induced BMP/MAPK signalling in vitro. a Representative Western blot analyses of MAPK signalling intermediates in sh-NC and sh-Mtdh cells. b, c Relative protein expression shown in (a). *P < 0.05 compared with sh-NC; #P < 0.05 compared with sh-NC + TGF-β1; ##P < 0.01 compared with sh-NC + TGF-β1. d Representative Western blot analyses of Smad1/5/9 and Smad2/3 phosphorylation in sh-NC and sh-Mtdh cells. e, f Relative protein expression shown in (d). *P < 0.05 compared with sh-NC; ** P < 0.01 compared with sh-NC; #P < 0.05 compared with sh-NC + TGF-β1; & P < 0.05 compared with sh-Mtdh; ns P > 0.05 compared with sh-NC + TGF-β1. g Representative Western blot analyses of BMPR1a in sh-NC and sh-Mtdh cells. h Relative protein expression shown in (g). ***P < 0.0001 compared with sh-NC; #P < 0.05 compared with sh-NC + TGF-β1. The data are presented as the mean ± SEM of at least three independent experiments