Gut Microbiome

Integrating WGCNA and network pharmacology to unravel the multi-target mechanisms of Ludangshen Oral liquid against nephrotic syndrome: Involvement of TGF-β1/Smad3 and gut microbiota.

TL;DR

LDSKFY exerts multi-target mechanisms against nephrotic syndrome involving the TGF-β1/Smad3 pathway and gut microbiota modulation, as validated through integrated WGCNA, network pharmacology, in vivo, and in vitro approaches.

Key Findings

UPLC-MS analysis identified 20 important active components in Ludangshen oral liquid (LDSKFY).

  • Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) was used to analyze the chemical composition of LDSKFY.
  • Network pharmacology predictions suggested that the mechanism of action of these components was associated with the TGF-β signaling pathway.
  • Molecular docking and dynamics simulations confirmed that multiple active components stably bound to key targets including TGF-β1, Smad3, and Smad7.

LDSKFY reduced kidney index and histopathological grading in adriamycin-induced nephrotic syndrome rats.

  • An Adriamycin (ADR)-induced NS model in SD rats was used as the in vivo model.
  • Renal histopathology was examined to assess morphological changes.
  • LDSKFY treatment resulted in reduced kidney index and improved histopathological grading compared to the ADR-induced NS group.

LDSKFY significantly improved multiple biochemical markers of renal function in ADR-induced nephrotic syndrome rats.

  • LDSKFY significantly decreased levels of 24-h urinary protein (24h-TP), 24-h microalbumin (24h-mALB), triglyceride (TG), cholesterol (CHO), blood urea nitrogen (BUN), and serum creatinine (CRE).
  • LDSKFY increased total protein (TP) and albumin (ALB) levels, indicating improved renal function.
  • These findings collectively indicate restoration of kidney function parameters toward normal values.

LDSKFY restored podocyte-specific marker expression and suppressed fibrosis markers in ADR-induced HBZY-1 cells in vitro.

  • An ADR-induced fibrosis model in HBZY-1 cells was used as the in vitro model.
  • LDSKFY effectively restored the expression of podocyte-specific markers nephrin and synaptopodin.
  • LDSKFY suppressed desmin expression, a marker associated with podocyte injury and fibrosis.
  • Western blot and RT-qPCR were used to assess protein and mRNA expression levels.

LDSKFY prevented activation of the TGF-β1/Smad3 signaling pathway in both renal tissue and HBZY-1 cells.

  • TGF-β1/Smad3 pathway-related protein and mRNA expression were assessed by Western blot and RT-qPCR in both in vivo and in vitro models.
  • The mechanism was predicted through an integrated approach combining WGCNA and network pharmacology prior to experimental validation.
  • Suppression of TGF-β1/Smad3 pathway activation was observed in both renal tissue from ADR-induced NS rats and in ADR-treated HBZY-1 cells.

LDSKFY treatment promoted a more stable gut microbial community structure in nephrotic syndrome rats.

  • 16S rRNA gene sequencing technology was used to investigate gut microbiota changes in rat fecal samples.
  • LDSKFY treatment was associated with promotion of a more stable gut microbial community structure compared to untreated NS model rats.
  • Gut microbiota modulation was identified as one of the multi-target mechanisms of LDSKFY against nephrotic syndrome.

Molecular docking and dynamics simulations confirmed stable binding interactions between multiple LDSKFY active components and key targets TGF-β1, Smad3, and Smad7.

  • Molecular docking was performed to validate interactions between active components identified by UPLC-MS and key targets predicted by network pharmacology.
  • Molecular dynamics simulations were used to further confirm the stability of these binding interactions.
  • Key targets validated included TGF-β1, Smad3, and Smad7, which are central components of the TGF-β signaling pathway.
  • These computational validations supported the mechanistic predictions derived from the WGCNA and network pharmacology integrated approach.

An integrated WGCNA and network pharmacology approach successfully predicted the TGF-β signaling pathway as the primary mechanism of LDSKFY against nephrotic syndrome.

  • Weighted gene co-expression network analysis (WGCNA) was combined with network pharmacology to predict potential mechanisms.
  • Network pharmacology predictions identified the TGF-β signaling pathway as being associated with the mechanism of action of LDSKFY active components.
  • These predictions were subsequently validated through in vivo, in vitro, and computational experiments.

What This Means

This research investigated how a traditional Chinese medicine product called Ludangshen oral liquid (LDSKFY), used clinically to treat chronic kidney inflammation, works against nephrotic syndrome — a serious kidney disorder characterized by excessive protein loss in urine, swelling, and impaired kidney function. The researchers used a combination of advanced chemical analysis, computer-based drug network predictions, animal experiments, cell culture experiments, and gut bacteria analysis to piece together how LDSKFY affects the disease at multiple levels. They identified 20 active chemical components in LDSKFY and found that these components appear to work primarily by blocking a biological signaling pathway called TGF-β1/Smad3, which is known to drive kidney scarring and damage. In rats with adriamycin-induced kidney disease, LDSKFY treatment improved multiple measures of kidney health, including reducing protein in the urine, normalizing cholesterol and blood lipid levels, and reducing markers of kidney failure like creatinine and urea nitrogen. In kidney cells grown in the laboratory, LDSKFY helped restore the normal architecture of specialized kidney filter cells (podocytes) and reduced signs of fibrosis (scarring). Beyond the kidney-specific effects, this research also suggests that LDSKFY influences the gut microbiome — the community of bacteria living in the intestines — by promoting a more stable bacterial community in treated animals. This finding points to a potential gut-kidney connection in how LDSKFY works. Computer modeling confirmed that several of LDSKFY's active chemical components can physically bind to and interact with the key proteins in the TGF-β1/Smad3 pathway (TGF-β1, Smad3, and Smad7), providing a molecular explanation for the observed biological effects. This research suggests that LDSKFY works through multiple simultaneous mechanisms — primarily by dampening harmful kidney fibrosis signals and also by reshaping gut bacterial communities — rather than through a single drug target. These findings provide a scientific framework for understanding why this traditional medicine may be effective for kidney disease and could support its broader clinical use for nephrotic syndrome and chronic kidney inflammation, though further clinical studies in humans would be needed to confirm these effects.

Have a question about this study?

Citation

Cheng Z, Xiang W, Chen X, Xu X, Hou W, Gao J, et al.. (2026). Integrating WGCNA and network pharmacology to unravel the multi-target mechanisms of Ludangshen Oral liquid against nephrotic syndrome: Involvement of TGF-β1/Smad3 and gut microbiota.. Fitoterapia. https://doi.org/10.1016/j.fitote.2026.107239