Geniposide Stabilized Atherosclerosis Plaque by Induced M2 Polarization via PPARγ Signaling Pathway [Letter]

Dear editor

We were very interested to read the article by Jin et al (2025) entitled “Geniposide Stabilized Atherosclerosis Plaque by Induced M2 Polarization via PPARγ Signaling Pathway” in Drug Design, Development and Therapy.¹ Through in vivo and in vitro experiments, the authors show that Geniposide (Gen) stabilizes atherosclerotic plaques by promoting macrophage M2 polarization via activation of the PPARγ signaling pathway. These findings offer valuable, albeit preliminary, insights into the anti-atherosclerotic actions of geniposide. However, we believe that several methodological aspects would benefit from greater clarification to enhance the mechanistic support for these various findings.

After carefully reading the manuscript, we feel that the reasons for choosing particular dose and treatment duration of the PPARγ antagonist GW9662 need further clarification. The in vitro studies were done using only one concentration (5 μM) and one pretreatment period (1 h), without testing whether the treatment affects cell viability under these conditions. Earlier findings suggest that GW9662 can be cytotoxic or exhibit off-target effects that are PPARγ non-inhibitory.² The downregulation of M2 marker expression may not only be due to specific PPARγ blockade, as it cannot be excluded that drug-induced effects on cellular state also caused this downregulation through nonspecific stress responses. In addition, GW9662 was given via intraperitoneal injection in the ApoE⁻/⁻ mouse model 1 mg/kg/day. Nevertheless, the regimen was not validated by any pharmacokinetic or pharmacodynamic data. Since GW9662 is known to have a poor in vivo bioavailability,³ and there is no information regarding systemic exposure, tissue distribution (in particular the aorta) or inhibition of the target pathway, it remains unclear whether this dose achieved effective and selective PPARγ blockade in vivo.

Moreover, it would be beneficial to strengthen the evidence for the necessity of PPARγ. Though GW9662 is commonly used as a selective PPARγ antagonist, it is prone to off-target effects with high concentrations or under specific experimental conditions, possibly affecting other nuclear receptors such as PPARα or PPARδ.^4,5 Accordingly, reliance on pharmacological inhibition alone may not fully rule out PPARγ-independent pathways. Using genetic methods like macrophage-specific PPARγ knockdown or knockout would significantly strengthen the conclusion that Gen acts mainly through PPARγ to promote M2 polarization.

The evaluation of plaque stability seems somewhat limited, and we observed a slight methodological flaw in the study. The study appears to equate reduced plaque area with enhanced stability. While plaque area is clinically relevant, stability is more strongly determined by key histopathological features of vulnerability—namely, fibrous-cap thickness, collagen content, and necrotic core size. The conclusion regarding plaque stabilization is therefore constrained by the absence of these critical parameters. In addition, the restriction enzyme listed as “HandIII” in the Methods section is presumably a typo for “HindIII.”

The authors should be commended for providing valuable preliminary evidence on the anti-atherosclerotic mechanism of Gen. Addressing these points through further clarification or additional experimentation would considerably reinforce the study’s conclusions and lay a firmer foundation for future research.