Saikosaponin b1 Attenuates Liver Fibrosis by Blocking STAT3/Gli1 Interaction and Inducing Gli1 Degradation

May combine two claims.

Activation of HSCs is the major source of ECM and the key driver of liver fibrosis.

“Activation of hepatic stellate cells (HSCs) is the major source of ECM and the key driver of liver fibrosis [5].”

• cites [5] V. Krizhanovsky, M. Yon, R. A. Dickins, et al., “ Senescence of Activated Stellate Cells Limits Liver Fibrosis,” Cell 134 (2008): 657– 667. 18724938 PMC3073300 sourceSupported

Classified as probably not a substantial claim. May combine two claims.

Quiescent HSCs are activated by profibrotic stimuli and transdifferentiated into activated HSCs, which produce excess Collagen I and facilitate ECM formation.

“Quiescent HSCs are activated by profibrotic stimuli and transdifferentiated into activated HSCs (aHSCs), which can produce excess Collagen I and facilitate ECM formation [6].”

• cites [6] E. Albanis and S. L. Friedman, “ Antifibrotic Agents for Liver Disease,” American Journal of Transplantation 6 (2006): 12– 19. 16433751 sourceSupported

May combine two claims.

Ssb1 can effectively drive the apoptosis of aHSCs and the degradation of ECM in CCl4-induced fibrotic models, thereby protecting mice against liver fibrosis.

“Our previous study demonstrated that Ssb1 can effectively drive the apoptosis of aHSCs and the degradation of ECM in CCl4‐induced fibrotic models, therefore protecting mice against liver fibrosis [17].”

• cites [17] M. Peng, M. Shao, H. Dong, et al., “ Nanodrug Rescues Liver Fibrosis via Synergistic Therapy With H2O2 Depletion and Saikosaponin b1 Sustained Release,” Communications Biology 6 (2023): 184. 36797395 PMC9935535 sourcePartly supported

  The Results section on cellular antifibrotic efficiency reports that Ssb1 induced apoptosis in activated HSCs. The mouse Results report reduced collagen-gene expression after Ssb1 treatment, but do not establish ECM degradation. The Discussion says Ssb1 alone was minimally effective at reversing fibrosis in CCl4-treated mice; the more effective treatment combined Ssb1 with MnO2. (LLM, conf=0.89)

  Experimental — accuracy not yet measured

May combine two claims.

aHSCs are the major producers of ECM components, which are key elements in liver fibrogenesis.

“aHSCs are known to be the major producers of ECM components, which are key elements in liver fibrogenesis [20].”

• cites [20] V. Carloni, R. G. Romanelli, M. Pinzani, G. Laffi, and P. Gentilini, “ Expression and Function of Integrin Receptors for Collagen and Laminin in Cultured Human Hepatic Stellate Cells,” Gastroenterology 110 (1996): 1127– 1136. 8613002 sourceSupported

May combine two claims.

STAT3 Tyr 705 phosphorylation induces STAT3 dimerization and nuclear translocation and upregulates STAT3's transcriptional activity.

“STAT3 Tyr 705 phosphorylation has been reported to induce STAT3 dimerization and nuclear translocation and upregulate STAT3's transcriptional activity [28, 29].”

• cites [28] X. Guan, J. Yang, W. Wang, et al., “ Dual Inhibition of MYC and SLC39A10 by a Novel Natural Product STAT3 Inhibitor Derived From Chaetomium Globosum Suppresses Tumor Growth and Metastasis in Gastric Cancer,” Pharmacological Research 189 (2023): 106703. 36804016 sourceNot checked — no full text of the source
• cites [29] J. Wang, Z. Huang, L. Ji, et al., “ SHF Acts as a Novel Tumor Suppressor in Glioblastoma Multiforme by Disrupting STAT3 Dimerization,” Advanced Science 9 (2022): e2200169. 35843865 PMC9475553 sourceSupported

Classified as probably not a substantial claim.

Increased tyrosine 705 phosphorylation of STAT3 in liver fibrosis can promote HSC proliferation and activation and contribute to liver fibrogenesis.

“Previous researchers have observed increased tyrosine 705 (Tyr 705) phosphorylation of STAT3 in liver fibrosis, which can promote HSC proliferation and activation and contribute to liver fibrogenesis [21, 22].”

• cites [21] Z. Hu, Y. Han, Y. Liu, et al., “ CREBZF as a Key Regulator of STAT3 Pathway in the Control of Liver Regeneration in Mice,” Hepatology 71 (2020): 1421– 1436. 31469186 sourceNot checked — no full text of the source
• cites [22] T.‐H. Su, C.‐W. Shiau, P. Jao, et al., “ Sorafenib and Its Derivative SC‐1 Exhibit Antifibrotic Effects Through Signal Transducer and Activator of Transcription 3 Inhibition,” Proceedings of the National Academy of Sciences of the United States of America 112 (2015): 7243– 7248. 26039995 PMC4466718 sourcePartly supported

  The ‘P-STAT3 Overexpression in CHB Patients with Advanced Liver Fibrosis’ section reports increased p-STAT3 in HSCs as fibrosis advances in patients with chronic hepatitis B. The Discussion states that STAT3 activation in HSCs stimulates their proliferation and activation, but the passages identify p-STAT3 without specifying phosphorylation at Tyr 705; the patient finding is also limited to chronic hepatitis B. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

May combine two claims.

Activated STAT3 can physically interact with Gli1 and lead to Gli1 enrichment in promoters of target genes controlled by Gli1-binding sites.

“Aside from the direct regulation of Gli1 expression, recent evidence indicates that activated STAT3 can physically interact with Gli1 and lead to Gli1 enrichment in the promoters of target genes controlled by Gli1‐binding sites [32, 35].”

• cites [32] S. R. Sirkisoon, R. L. Carpenter, T. Rimkus, et al., “ Interaction Between STAT3 and GLI1/tGLI1 Oncogenic Transcription Factors Promotes the Aggressiveness of Triple‐Negative Breast Cancers and HER2‐Enriched Breast Cancer,” Oncogene 37 (2018): 2502– 2514. 29449694 PMC5948110 sourcePartly supported

  In Results, ‘STAT3 forms complexes with GLI1 and tGLI1’ (page not available), immunoprecipitation shows a physical interaction, and ChIP shows GLI1 binding at GLI1-binding sites in the PTCH1 and GLI1 promoters. Co-expression with constitutively active STAT3 also enhances a GLI1-binding-site reporter. The passage does not show that activated STAT3 causes increased GLI1 enrichment at those promoters; it establishes promoter binding and enhanced reporter activity, not that causal change in occupancy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [35] R. M. Carr, P. A. Romecin Duran, E. J. Tolosa, et al., “ The Extracellular Sulfatase SULF2 Promotes Liver Tumorigenesis by Stimulating Assembly of a Promoter‐Looping GLI1‐STAT3 Transcriptional Complex,” Journal of Biological Chemistry 295 (2020): 2698– 2712. 31988246 PMC7049957 sourcePartly supported

  In the ‘SULF2 promotes liver cancer through GLI1-STAT3 complex’ section (page unavailable), co-immunoprecipitation supports a physical interaction between STAT3 and GLI1 after SULF2 expression. The promoter-binding results show GLI1 enrichment under SULF2 expression, but STAT3 knockdown did not significantly reduce GLI1 enrichment at GLI1-binding sites. Thus, the paper does not establish that activated STAT3 causes GLI1 enrichment at those sites. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

STAT3 binds to the promoter of the Gli1 gene and controls Gli1 expression.

“Recent reports showed that STAT3 binds to the promoter of the Gli1 gene and controls Gli1 expression [32, 33, 34].”

• cites [32] S. R. Sirkisoon, R. L. Carpenter, T. Rimkus, et al., “ Interaction Between STAT3 and GLI1/tGLI1 Oncogenic Transcription Factors Promotes the Aggressiveness of Triple‐Negative Breast Cancers and HER2‐Enriched Breast Cancer,” Oncogene 37 (2018): 2502– 2514. 29449694 PMC5948110 sourcePartly supported

  The Results section (‘STAT3 forms complexes with GLI1 and tGLI1,’ page not provided) reports ChIP evidence that STAT3 binds the GLI1 gene promoter. The expression results concern other genes, including Cep70 and UPF3A; the passages do not establish that STAT3 controls GLI1 expression. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [33] T. Manshouri, I. Veletic, Y. Zhang, et al., “ STAT3‐Induced Gli1 Promotes the Differentiation and Maturation of Myelofibrosis Fibrocytes,” Blood 134 (2019): 2961– 2961. sourceNot checked — no full text of the source
• cites [34] T. Manshouri, I. Veletic, P. Li, et al., “ GLI1 Activates Pro‐Fibrotic Pathways in Myelofibrosis Fibrocytes,” Cell Death & Disease 13 (2022): 481. 35595725 PMC9122946 sourceSupported

Classified as probably not a substantial claim.

Activated Gli1 translocates to the nucleus and activates transcription of genes that promote cell survival and proliferation, such as Gli1, Ptch1, and Bcl-2.

“Activated Gli1 translocates to the nucleus and activates the transcription of genes that promote cell survival and proliferation, such as Gli1, Ptch1, and Bcl‐2 [31].”

• cites [31] X. Gai, K. Tu, C. Li, Z. Lu, L. R. Roberts, and X. Zheng, “ Histone Acetyltransferase PCAF Accelerates Apoptosis by Repressing a GLI1/BCL2/BAX Axis in Hepatocellular Carcinoma,” Cell Death & Disease 6 (2015): e1712– e1712. 25855960 PMC4650545 sourcePartly supported

  The Results section reports GLI1 nuclear translocation, occupancy of the PTCH1 promoter, and changes in GLI1 and PTCH1 expression. The Abstract says GLI1 upregulates its own expression, while the Discussion links GLI1 to Bcl-2 regulation and resistance to apoptosis. Our copy does not establish that all three named genes promote both cell survival and proliferation. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Gli1 has three main functional domains including SUFU binding sites, DNA binding domain, and C-terminal transcriptional activation domain.

“Gli1 has three main functional domains including SUFU binding sites, DNA binding domain, and C‐terminal transcriptional activation domain [36].”

• cites [36] N. K. Agarwal, C. H. Kim, K. Kunkalla, et al., “ Active IKKβ Promotes the Stability of GLI1 Oncogene in Diffuse Large B‐Cell Lymphoma,” Blood 127 (2016): 605– 615. 26603838 PMC4742548 sourcePartly supported

  The Introduction (page not provided) says GLI1 has zinc-finger domains that bind DNA and a C-terminal transcriptional activation domain. It does not identify SUFU-binding sites or describe these as GLI1’s three main functional domains. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

STAT3 is a key transcriptional mediator of cellular processes.

“STAT3, a member of the STATs family, is a key transcriptional mediator of cellular processes [43, 44].”

• cites [43] V. Hernandez‐Gea and S. L. Friedman, “ Pathogenesis of Liver Fibrosis,” Annual Review of Pathology 6 (2011): 425– 456. 21073339 sourceNot checked — no full text of the source
• cites [44] H. Yu, D. Pardoll, and R. Jove, “ STATs in Cancer Inflammation and Immunity: A Leading Role for STAT3,” Nature Reviews Cancer 9 (2009): 798– 809. 19851315 PMC4856025 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Activated STAT3 can regulate transcription of many target genes involved in cell proliferation, migration, differentiation, and apoptosis.

“Activated STAT3 can regulate the transcription of many target genes involved in cell proliferation, migration, differentiation, and apoptosis [28, 45].”

• cites [28] X. Guan, J. Yang, W. Wang, et al., “ Dual Inhibition of MYC and SLC39A10 by a Novel Natural Product STAT3 Inhibitor Derived From Chaetomium Globosum Suppresses Tumor Growth and Metastasis in Gastric Cancer,” Pharmacological Research 189 (2023): 106703. 36804016 sourceNot checked — no full text of the source
• cites [45] A. K. Ajay, L. Zhao, S. Vig, et al., “ Deletion of STAT3 From Foxd1 Cell Population Protects Mice From Kidney Fibrosis by Inhibiting Pericytes Trans‐Differentiation and Migration,” Cell Reports 38 (2022): 110473. 35263586 PMC10027389 sourcePartly supported

  The Introduction says STAT3 initiates transcriptional programs, and the Results report effects of activated STAT3 on pericyte proliferation and migration. The Results also identify Collagen1a1 as a direct transcriptional target involved in fibrotic signaling. These passages do not establish the full claimed range of target genes, particularly genes involved in apoptosis. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim. May combine two claims.

CCD has a large hydrophilic surface predominantly involved in recruitment of STAT3 to its receptor, essential for activation via SH2 domain-mediated receptor binding, phosphorylation, and nuclear translocation.

“CCD has a large hydrophilic surface predominantly involved in the recruitment of STAT3 to its receptor [50], which is essential not only for its activation via SH2 domain‐mediated receptor binding but also for its activation via phosphorylation [51] and nuclear translocation [52].”

• cites [50] J. Dong, X. D. Cheng, W. D. Zhang, and J. J. Qin, “ Recent Update on Development of Small‐Molecule STAT3 Inhibitors for Cancer Therapy: From Phosphorylation Inhibition to Protein Degradation,” Journal of Medicinal Chemistry 64 (2021): 8884– 8915. 34170703 sourceNot checked — no full text of the source
• cites [51] Y. Wang, S. Wu, X. Zhu, et al., “ LncRNA‐Encoded Polypeptide ASRPS Inhibits Triple‐Negative Breast Cancer Angiogenesis,” Journal of Experimental Medicine 217 (2020): e20190950. PMC7062514 31816634 sourceSupported
• cites [52] J. Ma, T. Zhang, V. Novotny‐Diermayr, A. L. C. Tan, and X. Cao, “ A Novel Sequence in the Coiled‐Coil Domain of Stat3 Essential for Its Nuclear Translocation,” Journal of Biological Chemistry 278 (2003): 29252– 29260. 12746441 sourceNot checked — no full text of the source

May combine two claims.

Ssb1 is a promising antifibrosis compound effective in inducing aHSC apoptosis.

“Our previous study showed that Ssb1 is a promising antifibrosis compound that is effective in inducing aHSC apoptosis [17].”

• cites [17] M. Peng, M. Shao, H. Dong, et al., “ Nanodrug Rescues Liver Fibrosis via Synergistic Therapy With H2O2 Depletion and Saikosaponin b1 Sustained Release,” Communications Biology 6 (2023): 184. 36797395 PMC9935535 sourceSupported

May combine two claims.

Ssb1 inhibits STAT3 phosphorylation in activated HSC-T6 cells without influencing other possible binding proteins such as PI3K, AKT, and MAPK3.

“Our study revealed that Ssb1 inhibited STAT3 phosphorylation in the activated HSC‐T6 cells without influencing other possible binding proteins, such as PI3K, AKT, and MAPK3 [48, 49].”

• cites [48] N. Odagiri, T. Matsubara, M. Higuchi, et al., “ Involvement of ERK1/2 Activation in the Gene Expression of Senescence‐associated Secretory Factors in Human Hepatic Stellate Cells,” Molecular and Cellular Biochemistry 455 (2018): 7– 19. 30426301 sourceNot checked — no full text of the source
• cites [49] L. Wang, Y. Feng, X. Xie, et al., “ Neuropilin‐1 Aggravates Liver Cirrhosis by Promoting Angiogenesis via VEGFR2‐Dependent PI3K/Akt Pathway in Hepatic Sinusoidal Endothelial Cells,” EBioMedicine 43 (2019): 525– 536. 31060904 PMC6558257 sourceCould not verify

  The Results section on NRP-1 and VEGFR2-dependent angiogenesis reports PI3K/Akt phosphorylation in endothelial cells, not the effect of Ssb1 in activated HSC-T6 cells. Searches for Ssb1, STAT3, HSC-T6, and MAPK3 found no passage addressing the claimed inhibition or lack of effect on the other proteins. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

May combine two claims.

Bio-Ssb1 remarkably pulls down STAT3 protein in lysates from HSC-T6/LX-2 cells and mouse liver tissues, but no obvious interactions between JAK2 and Ssb1 or IL-6 and Ssb1 are observed.

“As indicated in Figure 3B–D, Bio‐Ssb1 remarkably pulled down the STAT3 protein in the lysates from cells and tissues, but no obvious interactions between JAK2 and Ssb1 or IL‐6 and Ssb1 were observed (Figure S3C,E).”

May combine two claims.

Different-concentration treatments of Ssb1 (0-10 μM) increase thermal stabilization of STAT3; 5 μM Ssb1 stabilizes STAT3 with a Tm shift of 3.43°C, suggesting intracellular binding of Ssb1 and STAT3.

“The isothermal dose‐response curve in Figure. 3G indicated that different‐concentration treatments of Ssb1 (0–10 µM) increased the thermal stabilization of STAT3. Moreover, 5 µM Ssb1 stabilized STAT3 with a T m shift of 3.43°C, suggesting the intracellular binding of Ssb1 and STAT3 (Figure 3H).”

May combine two claims.

Ssb1 inhibits fibronectin, collagen I, vimentin, α-SMA, and desmin expression in HSC-T6 cells in a dose-dependent manner, and 5 μM Ssb1 efficiently suppresses HSC activation.

“Western blot (WB) assay showed that Ssb1 inhibited fibronectin (FN), collagen I, vimentin, α‐SMA, and desmin expression in the HSC‐T6 cells in a dose‐dependent manner, and 5 µM Ssb1 could efficiently suppress HSC activation (Figure 1D; Figure S1D).”

May combine two claims.

The binding free energy of the Ssb1/STAT3-S319 complex is −49.28 kcal·mol−1, indicating strong binding.

“Binding free energy was evaluated using the MM/GBSA method, which revealed that the binding free energy of the Ssb1/STAT3‐S319 complex was −49.28 kcal·mol−1. This result indicates strong binding (Figure 4C).”

May combine two claims.

In liver cirrhosis tissue and CCl4-induced fibrotic liver of Balb/c mouse, STAT3 phosphorylation increases in α-SMA high-expressed fibrosis area, and obvious nucleus translocation of p-STAT3 is exhibited.

“Similar results were obtained from the liver cirrhosis tissue and CCl4‐induced fibrotic liver of Balb/c mouse. STAT3 phosphorylation increased in the α‐SMA high‐expressed fibrosis area, and obvious nucleus translocation of p‐STAT3 was exhibited (Figure S5B–E).”

May combine two claims.

In TGF-β1-stimulated HSC-T6 cells, STAT3 phosphorylation increases at 6 h, reaches maximum at 12 h, and is maintained up to 24 h.

“STAT3 phosphorylation increased at 6 h and reached the maximum level at 12 h; this level was maintained for up to 24 h, indicating that the activation of HSC‐T6 was accompanied by STAT3 Tyr 705 phosphorylation (Figure 5B).”

May combine two claims.

Wild-type STAT3 is pulled down by Bio-Ssb1, whereas S319A and R325A/D371A mutants, especially S319A, reduce the interaction of Bio-Ssb1 and decrease the amount of STAT3 proteins pulled down by Bio-Ssb1 in 293T cell lysates.

“The results showed that wild‐type STAT3 (WT) was pulled down by Bio‐Ssb1. However, the S319A and R325A/D371A mutants, especially S319A, reduced the interaction of Bio‐Ssb1, and a decreased amount of STAT3 proteins were pulled down by Bio‐Ssb1 in 293T cell lysates (Figure 4F).”

May combine two claims.

In STAT3-OE cells, Ssb1 treatment still inhibits STAT3 activation and reverses STAT3-mediated expression of fibrotic markers.

“In the STAT3‐OE cells, Ssb1 treatment still inhibited STAT3 activation and reversed the STAT3‐mediated expression of the fibrotic markers.”

May combine two claims.

Ssb1 binding inhibits STAT3 phosphorylation at Tyr 705 but does not influence Ser 727 phosphorylation in HSC-T6 and LX-2 cells.

“As shown in Figure 5C and Figure S7A,B, Ssb1 binding inhibited STAT3 phosphorylation in Tyr 705 but did not influence Ser 727 phosphorylation in the HSC‐T6 and LX‐2 cells.”

May combine two claims.

Ssb1 directly binds to STAT3 but not to upstream mediators JAK2 or IL-6.

“Collectively, these results confirmed that Ssb1 directly binds to STAT3 but not to upstream mediators.”

May combine two claims.

Among the Ssb1-binding proteins, STAT3 has the most remarkable binding and the highest IMean_Ratio.

“Among the proteins, STAT3 had the most remarkable binding and had the highest IMean_Ratio, indicating that Ssb1 was closely bound with STAT3.”

May combine two claims.

Bio-Ssb1-STAT3 binding in the protein microarray produced a robust SNR of 6.549, indicating Bio-Ssb1 could bind with STAT3 and label-free Ssb1 pretreatment effectively blocked Bio-Ssb1 binding.

“Bio‐Ssb1–STAT3 binding in the protein microarray produced a robust SNR of 6.549, indicating that Bio‐Ssb1 could bind with STAT3 and that label‐free Ssb1 pretreatment effectively blocked Bio‐Ssb1 binding (Figure 3A and Figure S3A).”

May combine two claims.

Compared with S3I-201, Ssb1 exhibits similar phosphorylation-inhibition efficiency but at a lower concentration, indicating a potent STAT3 inhibition capability.

“Compared with S3I‐201, Ssb1 exhibited similar phosphorylation‐inhibition efficiency but at a lower concentration, indicating a potent STAT3 inhibition capability (Figure 5D and Figure S7C).”

May combine two claims.

Ssb1 binding proteins are preferably enriched in the HIF-1 signaling pathway, and 14 Ssb1 directly binding proteins are involved in this pathway.

“The results showed that Ssb1 binding proteins were preferably enriched in the HIF‐1 signaling pathway (Figure 2D), and 14 Ssb1 directly binding proteins were involved in this pathway.”

May combine two claims.

STAT3 knockout decreases mRNA and protein expression of HSC activation biomarkers and collagen synthesis; conversely, STAT3 overexpression increases expression of fibrosis biomarkers and collagen synthesis.

“STAT3 KO decreased the mRNA and protein expression of HSC activation biomarkers and collagen synthesis. Conversely, overexpression of STAT3 (Figure S6E,F) increased the expression of fibrosis biomarkers and collagen synthesis.”

May combine two claims.

10 μM Ssb1 binding disrupts STAT3 dimerization effectively even in the presence of IL-6 and exerts an effect nearly equivalent to that of 50 μM S3I-201 on inhibiting STAT3 dimerization induced by IL-6.

“Moreover, 10 µM Ssb1 binding disrupted STAT3 dimerization effectively even in the presence of IL‐6 and exerted an effect that was nearly equivalent to that of 50 µM S3I‐201 on inhibiting STAT3 dimerization induced by IL‐6 (Figure 5E and Figure S7D).”

May combine two claims.

Ser319 is a peripheral modulatory residue and Arg325 is a direct binding residue around Site 2; Asp371 is near Site 3; no functionally relevant residues reported in literature are within 20 Å of Site 1 and Site 4.

“Ser319 (chain A, 18.82 Å) as a peripheral modulatory residue and Arg325 (chain A, 16.97 Å) as a direct binding residue of ligand were identified around Site 2, Asp371 (chain A, 11.67 Å) was identified near Site 3, and no functionally relevant residues reported in the literature were found within the 20 Å neighborhood of Site 1 and Site 4.”

May combine two claims.

Treatment with S3I-201 effectively inhibits phosphorylation of STAT3, reduces differentiation of HSCs into myofibroblasts, and inhibits collagen synthesis with decreased Collagen I level.

“Treatment with S3I‐201 effectively inhibited the phosphorylation of STAT3, reduced the differentiation of HSCs into myofibroblasts, and inhibited collagen synthesis with decreased Collagen I level (Figure S6A,B).”

May combine two claims.

When STAT3 expression is excluded, Tyr 705 phosphorylation and TGF-β1-induced expression of fibrotic markers decrease, and Ssb1 cannot further inhibit expression of Collagen I, α-SMA, and other fibrotic markers.

“When STAT3 expression was excluded, Tyr 705 phosphorylation and the TGF‐β1‐induced expression of fibrotic markers decreased. Ssb1 could not further inhibit the expression of Collagen I, α‐SMA, and other fibrotic markers (Figure S8E,F).”

May combine two claims.

Ssb1 can be effectively embedded within the STAT3-S319 pocket and forms multiple hydrogen-bonding interactions with key residues LYS244, HIS457, GLN326, and VAL323, suggesting higher affinity and stability compared with other pockets.

“Ssb1 could be effectively embedded within the STAT3‐S319 pocket and formed multiple hydrogen‐bonding interactions with the key residues, such as LYS244, HIS457, GLN326, and VAL323, suggesting that the binding of Ssb1 with S319 site of STAT3 showed higher affinity and stability compared with other pockets (Figure 4D,E; Figure S4A).”

May combine two claims.

Ssb1 decreases expression of TGF-β1-induced α-SMA and Collagen I, reducing TGF-β1-induced HSC activation and matrix protein expression.

“The results showed that Ssb1 decreased the expression of TGF‐β1‐induced α‐SMA and Collagen I and suggested that Ssb1 could reduce TGF‐β1‐induced HSC activation and matrix protein expression.”

May combine two claims.

After Ssb1 treatment, differentially expressed genes are remarkably enriched in the ECM-receptor interaction pathway, and many collagen genes are considerably decreased.

“Transcriptomic data revealed that differentially expressed genes were remarkably enriched in the ECM‐receptor interaction pathway after Ssb1 treatment, and many collagen genes were considerably decreased (Figure 1B).”

May combine two claims.

α-SMA level and phosphorylated STAT3 are elevated and observed in coincident areas with severity degree of fibrosis (F0-F4).

“The α‐SMA level and phosphorylated STAT3 were elevated and observed in coincident areas with the severity degree of fibrosis (F0–F4; Figure 5A).”

May combine two claims.

Ssb1 downregulates proinflammatory cytokines (IL-6 and IL-1β) and fibrotic proteins (FN, collagen I, α-SMA, and vimentin) in liver tissues.

“Meanwhile, the expression of various proinflammatory cytokines (IL‐6 and IL‐1β) and fibrotic proteins (FN, collagen I, α‐SMA, and vimentin) in the liver tissues was downregulated by Ssb1 treatment (Figure 9D; Figure S14C–E).”

May combine two claims.

Both Gli1 and STAT3 are co-localized in the nucleus in HSC cell lines, suggesting a functional correlation between the two proteins.

“IF double staining results showed that both Gli1 and STAT3 were co‐localized in the nucleus (Figure 7B), which suggested a functional correlation between the two proteins.”

May combine two claims.

10 mg·kg−1 Ssb1 exerts a considerable effect on fibrosis alleviation, about the same efficacy as S3I-201.

“Biomarkers of liver fibrosis, such as FN, Collagen I, desmin, and α‐SMA, were detected, and 10 mg·kg−1 Ssb1 exerted a considerable effect on fibrosis alleviation; which had about the same efficacy as the commercial inhibitor S3I‐201 (Figure 9A; Figure S13C).”

May combine two claims.

Ssb1 and S3I-201 reduce STAT3 phosphorylation and Gli1 expression in mouse liver.

“The WB results showed that Ssb1 and S3I‐201 reduced STAT3 phosphorylation and Gli1 expression in mouse liver (Figure 9B; Figure S13D).”

May combine two claims.

Gli1 overexpression increases Bcl2 expression without influencing STAT3 expression.

“Further, Gli1 overexpression along increased the expression of Bcl2 without influence the expression of STAT3 (Figure S11E).”

May combine two claims.

Ssb1 treatment induces MMP loss and mitochondrial dysfunction in HSC-T6 cells.

“indicating the MMP loss and mitochondrial dysfunction induced by Ssb1 treatment (Figure 8G).”

May combine two claims.

Ssb1 decreases p-STAT3 and Gli1 expression levels in the CCl4 model.

“The results in Figure 9E display an obvious decrease in p‐STAT3 and Gli1 expression levels.”

May combine two claims.

Ssb1 and S3I-201 decrease liver/body weight ratio and serum ALT, AST, and TBIL relative to untreated model group.

“Ssb1 and S3I‐201 decreased the liver/body weight ratio (liver index) and the serum levels of ALT, AST, and TBIL relative to those in the untreated model group (Figure S13B).”

May combine two claims.

Ssb1 treatment further downregulates Gli1 mRNA level, but no notable decrease of Gli1 protein expression is observed in Ssb1-treated STAT3-KO cells; by contrast, Ssb1 effectively decreases Gli1 level in STAT3-OE cells.

“Ssb1 treatment further downregulated Gli1 mRNA level, but no notable decrease of Gli1 protein expression was observed in the Ssb1 treated STAT3‐KO cells. By contrast, Ssb1 retained its inhibition compatibility and effectively decreased the level of Gli1 in the STAT3‐OE cells (Figure S9G,H).”

May combine two claims.

Ssb1 inhibits STAT3 phosphorylation and Gli1 expression in a dosage-dependent manner in HSC-T6 and LX-2 cells.

“The results of the HSC‐T6 and LX‐2 cells showed that Ssb1 inhibited STAT3 phosphorylation and Gli1 expression in a dosage‐dependent manner (Figure 6K and Figure S9C–E).”

May combine two claims.

Knockout or overexpression of STAT3 in HSC-T6 cells regulates expression of Gli1 at mRNA and protein levels, and Gli1 expression is dependent on STAT3.

“As presented in Figure 6G–J, knockout or overexpression of STAT3 in HSC‐T6 cells could regulate the expression of Gli1 at mRNA and protein levels, and Gli1 expression was dependent on STAT3 (Figure S9B).”

May combine two claims.

Ssb1 decreases STAT3 phosphorylation and Gli1 expression in aHSCs and blocks STAT3/Gli1 interaction in the CCl4 model.

“It was found that Ssb1 decreased STAT3 phosphorylation and Gli1 expression in the aHSCs and blocked the interaction between STAT3 and Gli1 (Figure S14F,G).”

May combine two claims.

Ssb1 and high-dose S3I-201 ameliorate TAA-induced morphological changes and liver damage with decreased collagen deposition.

“After 2 weeks of administration, we found that Ssb1 and high‐dose S3I‐201 remarkably ameliorated the TAA‐induced morphological changes and liver damage with decreased collagen deposition (Figure S13A).”

May combine two claims.

STAT3 is upstream of Gli1 and regulates the gene expression of Gli1.

“Our results showed that STAT3 was the upstream of Gli1 and regulated the gene expression of Gli1.”

May combine two claims.

Co-expression of p-STAT3 and Gli1 is increased by CCl4 induction and decreased by Ssb1 treatment in normal mice.

“Figure 9L indicates that the co‐expression of p‐STAT3 and Gli1 was increased by CCl4 induction and decreased by Ssb1 treatment in the normal mice.”

May combine two claims.

Ssb1 inhibits STAT3 activation in HSCs and attenuates liver fibrosis in a STAT3-dependent manner.

“Collectively, these results suggest that Ssb1 inhibited STAT3 activation in the HSCs and attenuated liver fibrosis in a STAT3‐dependent manner.”

May combine two claims.

Direct binding of Ssb1 successfully blocks STAT3 Tyr 705 phosphorylation, dimerization, and nuclear translocation.

“This observation suggests that direct binding of Ssb1 successfully blocked STAT3 Tyr 705 phosphorylation, dimerization, and nuclear translocation (Figure 5G and Figure S7F).”

May combine two claims.

Bio-Ssb1 retains the inhibition activity of fibrotic protein expression in activated HSC-T6 cells, and biotin modification does not influence the interaction between Ssb1 and proteins.

“Given that Bio‐Ssb1 retained the inhibition activity of fibrotic protein expression in the activated HSC‐T6 cells, biotin modification did not influence the interaction between Ssb1 and proteins (Figure 2C and S2A).”

May combine two claims.

STAT3-S319 may be the prominent site for Ssb1 selectively binding, and binding between Ssb1 and STAT3 further limits STAT3 phosphorylation.

“Collectively, these results demonstrate that STAT3‐S319 may be the prominent site for Ssb1 selectively binding, and the binding between Ssb1 and STAT3 further limited STAT3 phosphorylation.”

May combine two claims.

Compared with Ssa and Ssd, Ssb1 exerts less toxic side effects on normal hepatocytes, so it can alleviate liver diseases with no additional hepatoxicity.

“Compared with the widely studied Ssa and Ssd, Ssb1 exerts less toxic side effects on normal hepatocytes, so it can alleviate liver diseases with no additional hepatoxicity [13].”

• cites [13] S. Wang, Y. Zhang, Q. Zhang, et al., “ Content Decline of SERCA Inhibitors Saikosaponin A and D Attenuates Cardiotoxicity and Hepatotoxicity of Vinegar‐Baked Radix Bupleuri,” Environmental Toxicology and Pharmacology 52 (2017): 129– 137. 28412648 sourceNot checked — no full text of the source

May combine two claims.

Ssb1 reduces indices of liver fibrosis in TAA and CCl4-induced models and regulates STAT3 phosphorylation and p-STAT3/Gli1 interaction in fibrotic livers.

“Overall, the antifibrosis determination in animals showed that Ssb1 could reduce the indices of liver fibrosis in the TAA and CCl4‐induced models and regulate STAT3 phosphorylation and p‐STAT3/Gli1 interaction in fibrotic livers.”

Classified as probably not a substantial claim.

Gli1 is a downstream effector of the Hh signaling pathway.

“Gli1 is a downstream effector of the Hh signaling pathway [30].”

• cites [30] X. Zhu, S. Ye, D. Yu, et al., “ Physalin B Attenuates Liver Fibrosis via Suppressing LAP2α–HDAC1‐Mediated Deacetylation of the Transcription Factor GLI1 and Hepatic Stellate Cell Activation,” British Journal of Pharmacology 178 (2021): 3428– 3447. 33864382 sourceNot checked — no full text of the source

May combine two claims.

Ssb1-induced Bcl-2 downregulation induces Bax activation, MOMP, cytochrome c release, and intrinsic apoptosis.

“Therefore, the protein level of Bcl‐2 was downregulated with Ssb1 treatment, which induced the activation of Bax that results in MOM permeabilization (MOMP) and MOMP enables the release of cytochrome c released from the mitochondria into the cytoplasm which triggers the intrinsic or mitochondrial pathway of apoptosis.”

May combine two claims.

Ssb1 downregulates target genes and promotes apoptosis of activated HSCs.

“resulting in downregulation of target genes and promoting the apoptosis of activated HSCs.”

May combine two claims.

Direct binding of Ssb1 with STAT3 downregulates STAT3 transcriptional activity, thus regulating Gli1 expression and blocking the Hh signaling pathway.

“The luciferase activity of STAT3 was considerably reduced by 10 µM Ssb1, which meant that the direct binding of Ssb1 with STAT3 downregulated its transcriptional activity, thus regulating Gli1 expression and blocking the Hh signaling pathway (Figure 6M).”

May combine two claims.

Ssb1 decreases Gli1 nuclear import and transcriptional activity.

“and decrease Gli1 nuclear import and transcriptional activity”

May combine two claims.

STAT3 binds with Gli1 protein on the domain containing SUFU binding sites, competitively repressing binding of Gli1 and SUFU, thereby increasing Gli1 stability and nuclear translocation.

“Moreover, STAT3 could bind with Gli1 protein on the domain containing SUFU binding sites, which competitively repressed the binding of Gli1 and SUFU, therefore increasing Gli1 stability and nuclear translocation.”

May combine two claims.

Ssb1 represses interaction between STAT3 and Gli1, promoting Gli1 degradation by the ubiquitin-proteasome system.

“A series of examinations showed that Ssb1 repressed interaction between STAT3 and Gli1, which subsequently promoted Gli1 degradation by the ubiquitin‐proteasome system.”

May combine two claims.

Ssb1 reduces STAT3 tyrosine phosphorylation and prevents dimerization and nuclear translocation possibly because Ssb1 directly targets the S319 residues in CCD.

“In our research, Ssb1 reduced STAT3 tyrosine phosphorylation and prevented dimerization and nuclear translocation possibly because Ssb1 directly targeted the S319 residues in CCD.”

May combine two claims.

Gli1 degradation regulates Bcl2 expression in the Hh signaling pathway and induces aHSCs apoptosis through the mitochondrial pathway.

“As a result, Gli1 degradation regulated the expression of Bcl2 in the Hh signaling pathway and therefore induced aHSCs apoptosis through the mitochondrial pathway (Figure 8E).”

May combine two claims.

Ssb1 competitively prevents STAT3's interaction with Gli1, inhibiting its transcriptional activity and blocking Hh signaling activation.

“Meanwhile, Ssb1 competitively prevented STAT3's interaction with Gli1, which inhibited its transcriptional activity and blocked the activation of the Hh signaling pathway.”

Probably not a substantial claim: may not pass S3 (load-bearing).

This work did not evaluate STAT3 absence in macrophages and hepatocytes in the liver.

“This work did not evaluate STAT3 absence in macrophages and hepatocytes in the liver, although STAT3 is a key protein in inflammation.”

May combine two claims.

Ssb1 abolishes Gli1 function by directly binding with STAT3 and decreases Gli1 expression at gene and protein levels, resulting in loss of antiapoptotic mediators and inducing aHSCs apoptosis.

“Collectively, Ssb1 abolished Gli1 function by directly binding with STAT3 and decreased Gli1 expression both at the gene level and protein level, resulting in the loss of antiapoptotic mediators and inducing aHSCs apoptosis.”

May combine two claims.

Ssb1 binding with STAT3 inhibits transcriptional activity of STAT3, decreasing gene expression of Gli1.

“On the one side, Ssb1 binding with STAT3 inhibited the transcriptional activity of STAT3, which decreased the gene expression of Gli1.”

May combine two claims.

Ssb1 directly binds to STAT3, suppresses STAT3 activation, and induces aHSCs apoptosis by regulating the STAT3/Gli1/Bcl2 axis in the Hh signaling pathway.

“Ssb1 directly binds to STAT3, suppresses the activation of STAT3, and induces aHSCs apoptosis by regulating the STAT3/Gli1/Bcl2 axis in the Hh signaling pathway.”

Probably not a substantial claim: may not pass S3 (load-bearing).

STAT3 has six common structural motifs: NTD, CCD, DBD, linker domain, SH2, and transactivation domain.

“STAT3 has six common structural motifs, namely, NTD, CCD, DBD, the linker domain, the Src homology 2 domain (SH2), and the transactivation domain [50].”

• cites [50] J. Dong, X. D. Cheng, W. D. Zhang, and J. J. Qin, “ Recent Update on Development of Small‐Molecule STAT3 Inhibitors for Cancer Therapy: From Phosphorylation Inhibition to Protein Degradation,” Journal of Medicinal Chemistry 64 (2021): 8884– 8915. 34170703 sourceNot checked — no full text of the source

May combine two claims.

Ssb1 prevents liver fibrogenesis in TAA and CCl4-induced animal models by suppressing STAT3 activation and blocking STAT3/Gli1 interaction.

“Ssb1 prevented liver fibrogenesis in the TAA and CCl4‐induced animal models by suppressing STAT3 activation and blocking STAT3/Gli1 interaction”

May combine two claims.

Ssb1 binding with STAT3 blocks interaction between STAT3 and Gli1, promoting Gli1 binding with SUFU and impairing Gli1 protein stability by the ubiquitin-proteasome system.

“On the other hand, Ssb1 binding with STAT3 blocked the interaction between STAT3 and Gli1, which promoted Gli1 binding with SUFU and then impaired Gli1 protein stability by the ubiquitin‐proteasome system.”