Cajanonic acid A derivative XJ-60 improves liver fibrosis in mice with non-alcoholic fatty liver disease by inhibiting the SP1/TGF-β/Smad3 signaling axis

doi:10.12122/j.issn.1673-4254.2026.02.14

Abstract

Abstract:

Objective To investigate the mechanism of XJ-60, a derivative of cajanonic acid A, for alleviating liver fibrosis in mice with non-alcoholic fatty liver disease (NAFLD). Methods Twelve db/db mice were randomized equally into NAFLD model group and XJ-60 treatment group with daily gavage of 50 mg/kg XJ-60, with 6 db/m mice serving as the control group. Successful modeling of NAFLD was validated using HE and Oil Red O staining and by measuring ALT and AST levels of the mice. Immunohistochemistry was used for detecting hepatic expressions of specific protein 1 (SP1) and fibronectin (FN); the changes in hepatic expressions of epithelial-mesenchymal transition (EMT) markers, extracellular matrix (ECM) markers, and IL-6 were detected using Western blotting and ELISA. In AML12 cells induced with free fatty acids (FFA), the effect of XJ-60 on expressions of SP1, transforming growth factor-β (TGF-β), Smad3/p-Smad3, and the markers of EMT and ECM were analyzed. SP1 knockdown experiment was performed to validate the role of TGF‑β/Smad3 signaling axis in NAFLD. Results XJ-60 treatment significantly reduced serum lipid levels, improved liver histology, and lowered hepatic IL-6 expression in db/db mice. The expression level of SP1 was positively correlated with α-SMA and negatively with E-ca expression. In AML12 cells, XJ-60 treatment at 10 μmol/L significantly reduced FFA-induced lipid accumulation and downregulated cellular expressions of SP1, TGF-β, p-Smad3, and the EMT and ECM markers; SP1 knockdown obviously inhibited the expression levels of TGF-β, Smad3 and ECM markers. Conclusion XJ-60 ameliorates liver fibrosis and steatosis in mice with NAFLD possibly through SP1-mediated inhibition of the TGF‑β/Smad3 pathway to reduce ECM deposition.

Key words: non-alcoholic fatty liver disease, specific protein 1, transforming growth factor?β, Smad3 protein, cajanonic acid A derivative

Zhaodie GENG, Li HU, Yunli DAI, Ronggang LUO, Tao XU, Xuyang LIAO, Zhiping YUAN, Jianta WANG, Ying XIAO. Cajanonic acid A derivative XJ-60 improves liver fibrosis in mice with non-alcoholic fatty liver disease by inhibiting the SP1/TGF-β/Smad3 signaling axis[J]. Journal of Southern Medical University, 2026, 46(2): 362-373.

Figures/Tables 13

Tab.1 Primer sequence for RT-qPCR

Primer	Sequence(5´→3´)	Optimal Annealing Te-mperature (℃)
SP1	F: TGCCAAATCTACTCCAGCA	53.6
	R: AGCAAGGTGATGTTCCCA
FN	F: ATGTGGACCCCTCCTGATAGT	57.3
	R: GCCCAGTGATTTCAGCAAAGG
Col-IV	F: TTTGGCTCGCCACCATAGAG	60.04
	R: GCAGAGGCGAGCATCATAGT
TGF-β	F: CTCCCGTGGCTTCTAGTGC	57.2
	R: GCCTTAGTTTGGACAGGATCTG
E-ca	F:CAGGTCTCCTCATGGCTTTGC	57.7
	R:CTTCCGAAAAGAAGGCTGTCC
α-SMA	F: CCCAGACATCAGGGAGTAATGG	56.1
	R: TCTATCGGATACTTCAGCGTCA
GAPDH	F: GGTTGTCTCCTGCGACTTCA	57
	R: TGGTCCAGGGTTTCTTACTCC

Fig.1 Levels of blood glucose, triglycerides and total cholesterol and expression levels of ALT and AST in the liver tissue of the mice in each group (n=6, Mean±SD). A: Levels of blood glucose, triglyceride and total cholesterol in different groups. B: Expression levels of ALT and AST in the liver tissue in different groups. *P<0.05 vs NC; #P<0.05 vs NAFLD.

Fig.2 Pathological changes and fat accumulation in the liver tissue of the mice in each group (n=6, Mean±SD). A: HE staining. B: Oil red O staining.

Fig.3 Expression of IL-6 in the liver tissues of the mice in each group (n=6, Mean±SD). *P<0.05 vs NC; #P<0.05 vs NAFLD.

Fig.4 Protein levels of Sp1 and FN in the liver tissues of the mice in each group determined by Western blotting (n=6, Mean±SD). A, B: Western blotting for detecting protein expression levels of FN and SP1. C: RT-qPCR for detecting expressions of Sp1 and FN mRNA. D: Immunohistochemical staining for Sp1 and FN in the liver tissues of the mice in each group. *P<0.05 vs NC; #P<0.05 vs NAFLD.

Fig.5 Expression levels of Col-IV, E-ca and α-SMA in the liver tissues of the mice in each group detected by Western blotting (n=6, Mean±SD). *P<0.05 vs NC; #P<0.05 vs NAFLD.

Tab.2 Spearman rank correlation analysis

Protein/Marker	Sp1
Sp1	1.000
E-ca	-0.767**
α-SMA	0.777**

Fig.6 Comparison of cell survival rate of each group in CCK8 experiment (n=3, Mean±SD). A: Screening the safe concentration range of XJ-60 acting on AML12 cells without FFA intervention, P>0.05 vs NC. B: The optimal therapeutic concentration of XJ-60 acting on AML12 cells under FFA intervention. **P<0.01 vs PO.

Fig.7 　　Accumulation of lipid droplets in the hepatocytesin each group. A: Pathological changes and lipidaccumulation in hepatocytes of different groupsobserved using Oil Red O staining. B: Quantitativeanalysis of lipid accumulation in the hepatocytes in eachgroup (n=3, Mean±SD), **P<0.01 vs NC; ##P<0.01 vs PO.

Fig.8 Effect of XJ-60 on expressions of SP1/TGF-β/Smad3 signaling pathway in FFA-induced AML12 cells (n=3, Mean±SD). A, B: Protein expression levels of SP1, TGF-β and p-Smad3 in AML12 cells in each group (*P<0.05, **P<0.01 vs PO+DMSO). C: mRNA expression levels of SP1, TGF-β and p-Smad3 in hepatocytes of each group (**P<0.01,***P<0.001 vs PO+DMSO). D: SP1 and p-Smad3 immunofluorescence staining of AML12 cells in each group (×1000).

Fig.9 Changes in expressions of the protein markers in AML12 cells after SP1 knockdown (n=3, Mean±SD). A, B: Protein expression levels of FN, Col-IV, SP1, TGF‑β and p-Smad3 in AML12 cells (*P<0.05, **P<0.01 vs PO+Neg). C: mRNA expression levels of FN, Col-IV, SP1, TGF-β and p-Smad3 in AML12 cells in each group (*P<0.05, **P<0.01 vs PO+Neg).

Fig.10 Effect of XJ-60 treatment on expressions of EMT and ECM markers in AML12 cells (n=3, Mean±SD). A, B: Protein expression levels of FN, Col-IV, E-ca and α-SMA in AML12 cells in each group (*P<0.05, **P<0.01, ***P<0.001 vs PO+DMSO). C: mRNA expression levels of FN, Col-IV, E-ca and α-SMA in hepatocytes of each group (**P<0.01 vs PO+DMSO).

Fig.11 Effects of CAA and XJ-60 for improving fibrosis in AML12 cells (n=3, Mean±SD). A: Protein bands of FN, Col-IV and α-SMA in AML12 cells in each group. B: Relative protein expression levels. **P<0.01, ****P<0.0001 vs PO+CAA.

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