The Concentration Paradox: When Dose-Response Logic Collapses in a Photoaging Study [Response to Letter]

Dear editor

We sincerely thank Dr. Xiao and Dr. Tan for their careful reading of our research article entitled “Protective Effect of Idebenone Against UVB-Induced Photoaging in HaCaT Cells” and for raising critical questions regarding several limitations of our work.1,2 We have thoroughly reviewed all the comments and greatly appreciate the opportunity to provide clarification and further explanation. Below, we respond point-by-point to the raised concerns.

A Direct Logical Contradiction Exists in the Wound Healing Assay Results

We acknowledge that this was a writing error that occurred during manuscript preparation, rather than a data inversion or statistical mistake. We would like to clarify the experimental results as follows: three concentration gradients (0.5 nM, 1 nM, and 5 nM) were established in this study, and cell migration capacity increased progressively with rising drug concentrations, with the order of efficacy being 5 nM > 1 nM > 0.5 nM, thus identifying 5 nM as the optimal concentration. The original text inadvertently misstated the optimal concentration as 0.5 nM, resulting in confusion. We appreciate for their meticulous scrutiny in identifying this oversight.

Invalid Statistical Approach to Multiple Comparisons

We fully agree that testing over 30 biological endpoints via one-way ANOVA with Tukey’s post-hoc test without multiple comparison correction may have inflated Type I error rates. It should be noted that all measured readouts form a unified mechanistic cascade—UVB-triggered oxidative stress, mitochondrial damage, and skin photoaging—with tightly interconnected upstream and downstream readouts rather than fully independent screening metrics. Consistent expression trends across orthogonal assays (CCK-8, immunofluorescence, ELISA, Western blot, and RNA-seq) create mutually supportive evidence that partially mitigates false-positive risks from repeated testing. For readouts with broad error bars, such as SOD, we only presented raw data and statistical outcomes without overinterpreting subtle biological differences. Nevertheless, we fully recognize the importance of applying more rigorous statistical standards in multiple comparisons and will proactively refine our statistical analysis plan in future studies by incorporating multiple testing correction procedures to further ensure the robustness of our conclusions.

RNA-Seq Sample Size (n=3)

We recognize the concern that three biological replicates per group, while meeting minimum analytical standards, may limit differential expression reliability, especially for our IL-3RA and TUBA8 mechanistic conclusions. In this context, this study employed the HaCaT immortalized cell line, which exhibits high homogeneity and stability, with substantially lower sample heterogeneity than primary cells or animal tissues. Moreover, n=3 replicates are widely adopted in published exploratory in vitro transcriptomic work, which informed our sample design. We fully acknowledge that 4–6 replicates represent the field’s optimal standard and candidly admit that our design did not reach this level. We also agree that small replicate sets are more susceptible to outliers and technical variability. To reduce bias, we implemented strict sequencing quality control, outlier removal, depth normalization, and stringent cutoffs (|log₂FC| ≥ 1, P < 0.05) to minimize technical deviations. Moreover, the key targets IL-3RA and TUBA8 were independently validated by Western blot, corroborating their expression changes at the protein level and partially offsetting the limitation of transcriptomic sample size. Importantly, all mechanistic interpretations of these genes are restricted exclusively to our in vitro UVB-induced HaCaT photoaging model without overgeneralization.

Apparent Discrepancy Between Oxidative Stress-Mitochondrial Dysfunction Hypothesis and UVB Dose Data

Our statement that “oxidative stress often leads to mitochondrial dysfunction” was not intended to imply that mild or transient oxidative stress would immediately cause mitochondrial membrane potential (MMP) collapse; rather, the two are not linearly coupled. Mitochondria possess intrinsic antioxidant buffering systems and exhibit a tolerance threshold. Low-to-moderate UVB doses (10 and 20 mJ/cm²), although capable of reducing SOD activity and increasing MDA content, remain within mitochondrial compensatory capacity, thus maintaining MMP retention above 90% without overt depolarization. Only when the UVB dose reaches 30 mJ/cm² does oxidative stress exceed this compensatory limit, allowing accumulated ROS to disrupt mitochondrial membrane architecture and induce significant MMP decline. In brief, oxidative stress represents an early, sensitive damage marker, whereas MMP collapse constitutes a late-stage consequence of cumulative oxidative injury. The original phrasing—“oxidative stress often leads to mitochondrial dysfunction”—was overly generalized, failing to delineate the degree of insult or specify the compensatory threshold, and thus gave rise to logical misinterpretation. This constitutes a writing oversight on our part, for which we sincerely apologize.

Finally, we sincerely thank Dr. Xiao and Dr. Tan for their insightful comments and the journal for providing us with the opportunity to respond. Their suggestions are of great value in improving our research and enhancing the quality of the manuscript. We will carefully consider these recommendations in our future studies, and continue to provide more reliable evidence on the photoprotective effects of idebenone.

Disclosure

The author reports no conflicts of interest in this communication.