Cardiospermum halicacabum Extract Attenuates UVB-Induced Photoaging in Human Skin Fibroblasts via Inhibition of the PI3K/Akt/mTOR Signaling Pathway

TL;DR

This study shows that Cardiospermum halicacabum extract protects human skin cells from UVB-induced aging by reducing oxidative stress and targeting a key signaling pathway.

Contribution

The study identifies the PI3K/Akt/mTOR pathway as a novel target for CHE in mitigating UVB-induced photoaging.

Findings

CHE reduces UVB-induced cell senescence by downregulating p53 and p16 markers.

CHE inhibits ROS production and restores mitochondrial function in human skin fibroblasts.

CHE suppresses UVB-induced hyperactivation of the PI3K/Akt/mTOR signaling pathway.

Abstract

Solar ultraviolet B (UVB) irradiation is a primary environmental driver of skin photoaging, characterized by oxidative stress accumulation and mitochondrial dysfunction. In this study, we investigated the protective efficacy and underlying molecular mechanisms of Cardiospermum halicacabum extract (CHE) against UVB-induced senescence in human skin fibroblasts (HSFs). Phytochemical profiling via LC-MS characterized CHE as a rich source of bioactive flavonoids, organic acids, and glycosides. We demonstrated that pretreatment with CHE significantly ameliorated UVB-triggered cellular senescence, as evidenced by the alleviation of cell cycle arrest and the downregulation of senescence-associated markers p53 and p16. Furthermore, CHE effectively inhibited intracellular ROS generation and restored mitochondrial respiratory function. Transcriptomic analysis, validated by molecular assays, revealed that CHE exerts its protective effects primarily by suppressing the UVB-induced hyperactivation of the PI3K/Akt/mTOR signaling cascade. Collectively, these preliminary in vitro findings highlight CHE as a promising natural protective candidate that mitigates skin photoaging by targeting the PI3K/Akt/mTOR axis to attenuate oxidative stress and restore mitochondrial homeostasis.

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1. Introduction

The skin serves as the primary interface between the human body and the external environment, acting as a robust barrier against physical and biological insults. Skin aging is generally classified into two distinct processes: intrinsic (chronological) aging and extrinsic aging [1]. While the former is an inevitable physiological process governed by the passage of time and genetics, the latter is driven by external environmental stressors, including cigarette smoke, pollution, and solar radiation [2]. Among these factors, ultraviolet (UV) exposure is universally recognized as the predominant etiological factor in extrinsic aging, clinically termed “photoaging” [3,4]. Although the solar UV spectrum comprises UVA (320–400 nm), UVB (280–320 nm), and UVC (200–280 nm), UVB radiation is widely considered the most biologically damaging component reaching the earth’s surface [5]. Even though UVB constitutes a minor fraction of total solar energy (approximately 5%) compared to UVA, its high photon energy renders it more than 1000 times more erythemogenic than UVA, making it the primary driver of sunburn and photo-oxidative damage [6]. At the cellular level, the deleterious effects of UVB are largely orchestrated by the generation of reactive oxygen species (ROS). However, excessive UVB irradiation disrupts redox homeostasis, triggering a surge in ROS that can overwhelm endogenous antioxidant defenses, leading to a state of oxidative stress [7]. This imbalance not only causes direct damage to macromolecules but also aberrantly activates downstream signaling cascades, resulting in the upregulation of matrix metalloproteinases (MMPs). The subsequent degradation of collagen and elastin fibers in the extracellular matrix constitutes the structural hallmark of photoaged skin [8]. Although synthetic antioxidants (e.g., BHA and BHT) have been employed to counteract oxidative stress, their long-term application is often restricted by potential instability and adverse toxicological effects [9]. In light of these limitations, the identification of potent exogenous antioxidants from botanical sources—distinguished by their favorable safety profiles and multi-target efficacy—has emerged as a promising protective strategy to restore redox homeostasis and mitigate photoaging [10]. Recently, the application of metabolomics and systems pharmacology has significantly advanced this field by enabling the comprehensive mapping of plant-derived antioxidant networks [11].

Cardiospermum halicacabum L. (Sapindaceae), commonly known as the balloon vine, is a perennial climber widely distributed in tropical and subtropical regions of Asia and Africa [12]. In traditional medicine systems, particularly in India, it has been historically utilized for treating inflammatory conditions such as rheumatism, nervous disorders, and orchitis. This extensive ethnobotanical heritage, particularly within the Indian subcontinent, has catalyzed a substantial body of contemporary research, definitively establishing C. halicacabum as a vital natural repository of bioactive phytochemicals with pleiotropic pharmacological properties [13]. Phytochemical investigations have revealed that C. halicacabum is a rich reservoir of bioactive compounds, including flavonoids such as quercetin, and phenolic acids notably caffeic acid and chlorogenic acid, alongside other derivatives like coumarin [14]. Previous pharmacological studies have demonstrated its diverse bioactivities, ranging from free radical scavenging and anti-inflammatory effects to the inhibition of collagenolytic enzymes [15,16]. Consistent with recent phytochemical consensus, such bioactive flavonoids, particularly quercetin derivatives, act as primary effectors in neutralizing reactive oxygen species and conferring cytoprotection [17]. Despite its documented medicinal value, the specific potential of C. halicacabum extract (CHE) in dermatology, particularly regarding UVB-induced photoaging, remains underexplored. Moreover, the underlying molecular mechanisms—specifically whether CHE exerts its cytoprotective effects by modulating the PI3K/Akt/mTOR signaling pathway—have not been elucidated. This study undertakes a comprehensive investigation into the protective efficacy of CHE against UVB-induced senescence in human skin fibroblasts (HSFs). By integrating phytochemical profiling with mechanistic assays, we aim to validate the in vitro photoprotective efficacy of CHE as a novel candidate for mitigating skin photoaging.

2. Results

2.1. Phytochemical Characterization and Quantitative Analysis of C. halicacabum Extract

To comprehensively elucidate the chemical profile of CHE, we initially performed a qualitative analysis using UPLC-Orbitrap-MS. As illustrated in the base peak chromatogram (Figure 1A), a total of 31 putative compounds were identified by comparing their specific m/z values and fragmentation patterns against online mass spectral databases in both positive and negative ion modes. The identified constituents were predominantly flavonoids (including rutin, kaempferol, and myricitrin), alongside minor fractions of organic acids and glycosides (Table 1 and Table 2).

To further validate these findings and standardize the extract, we conducted quantitative analysis of key marker flavonoids using HPLC (Agilent 1200, Figure 1B). Quantitative determination revealed that CHE contains significant concentrations of bioactive flavonoids. Specifically, the contents of myricitrin, luteolin, and apigenin were determined to be 3297.17 ± 26.91 μg/g, 973.51 ± 7.24 μg/g, and 2820.64 ± 61.55 μg/g, respectively. These results confirm that CHE is a flavonoid-rich botanical source, establishing a reproducible chemical fingerprint for subsequent biological assays.

2.2. In Vitro Antioxidant and Reducing Potential of C. halicacabum Extract

To evaluate the direct antioxidant capacity of CHE, we employed FRAP, ABTS, and DPPH assays. First, the FRAP assay (Figure 2A) was utilized to assess the ferric reducing power of the extract. Results indicated that CHE functions as a potent electron donor, exhibiting a clear dose-dependent increase in reducing capacity, although the overall potency was slightly lower than that of the positive control, L-ascorbic acid.

Subsequently, we evaluated the free radical scavenging capabilities of CHE. As shown in Figure 2B,C, CHE exhibited a robust scavenging capacity against both ABTS and DPPH radicals, also in a dose-dependent manner. Specifically, for the DPPH assay (Figure 2C), the maximal scavenging rate was observed at a concentration of 2.5 mg/mL. Collectively, these non-cellular assays confirm that CHE possesses significant antioxidant potential through both electron donation and radical scavenging mechanisms. To specifically evaluate its biological relevance and safety in a physiological context, we proceeded to validate these findings using in vitro cell models.

2.3. CHE Protects HSFs Against UVB-Induced Cytotoxicity and Mitochondrial Dysfunction

To establish an optimal photoaging model, HSF cells were exposed to UVB irradiation for varying durations. We determined that an exposure of 60 s (corresponding to a dose of 48 mJ/cm^2^ at a distance of 15 cm) resulted in a significant reduction in cell viability to approximately 50% of the control group (Figure 3A). Consequently, this exposure condition (IC50 dose) was established as the standard modeling protocol. Prior to evaluating cytoprotection, we assessed the biocompatibility of CHE. As shown in Figure 3B, CHE exhibited a favorable safety profile, showing no cytotoxicity while promoting cell proliferation in a dose-dependent manner. Importantly, pretreatment with CHE significantly ameliorated UVB-induced cytotoxicity, with maximal protective effects observed at a concentration of 100 μg/mL (Figure 3C).

Further investigation into the mechanism focused on oxidative stress status. Flow cytometry analysis using the DCFH-DA probe revealed that UVB irradiation caused a distinct rightward shift in fluorescence intensity, indicating a massive accumulation of intracellular ROS (Figure 3D). However, CHE pretreatment effectively reversed this shift. Quantitative analysis confirmed that CHE dose-dependently mitigated the ROS surge, with the 100 μg/mL concentration showing efficacy comparable to the positive control (VE) (Figure 3E). These findings were visually corroborated by fluorescence microscopy, where the UVB-induced intense green fluorescence was substantially quenched by CHE treatment (Figure 3F,G), collectively demonstrating its potent capability to alleviate oxidative stress.

Given that excessive ROS accumulation precipitates mitochondrial impairment, we further examined the impact of CHE on mitochondrial bioenergetics. Crucially, CHE treatment effectively reversed UVB-induced mitochondrial dysfunction. As evidenced by the analysis of mitochondrial respiratory parameters (Figure 4A–E), CHE administration significantly restored basal respiration and ATP production, while enhancing spare respiratory capacity and attenuating proton leak. These data confirm that CHE exerts its protective effects by maintaining bioenergetic homeostasis and preventing oxidative damage.

2.4. CHE Alleviates UVB-Induced Cell Cycle Arrest and Senescence

To elucidate the mechanism underlying the cytoprotective effect of CHE, we analyzed cell cycle distribution using flow cytometry. As illustrated in Figure 5A,B, HSF cells under normal conditions predominantly resided in the G0/G1 phase. However, UVB irradiation significantly disrupted cell cycle progression, triggering a prominent S-phase arrest. Specifically, UVB exposure resulted in an approximately 20% increase in the S-phase population compared to the control group, indicating a blockage in DNA replication that impeded transition into the G2/M phase. Notably, pretreatment with CHE effectively relieved this blockade, significantly reducing the S-phase fraction by approximately 10% and restoring the cell cycle profile towards the control state.

To corroborate these findings at the molecular level, we examined the expression of key senescence and stress-associated proteins. Consistent with the cell cycle arrest, Western blot analysis revealed that UVB exposure elicited a robust DNA damage response, evidenced by the significant upregulation of p16 and the hyperphosphorylation of p53 (p-p53) (Figure 5C–E). Crucially, CHE treatment attenuated these pathological alterations in a concentration-dependent manner, effectively downregulating p16 expression and decreasing the p-p53/p53 ratio. These results suggest that CHE mitigates UVB-induced cellular senescence and DNA damage responses.

2.5. Transcriptomic Profiling Identifies PI3K/Akt Signaling as a Key Target of CHE

To systematically elucidate the molecular mechanisms underlying the cytoprotective effects of CHE, high-throughput RNA sequencing (RNA-seq) was performed. Differential expression analysis was conducted to identify significantly modulated genes. As visualized in the volcano plot (Figure 6A), CHE treatment significantly altered the transcriptomic landscape compared to the UVB-irradiated model group, with 31 genes upregulated and 232 genes downregulated (p < 0.05, |log2FC| > 1).

To identify potential therapeutic targets specifically associated with the protective effect of CHE, we performed an intersection analysis using a Venn diagram (Figure 6B). This comparison revealed 138 overlapping genes, representing critical targets where CHE effectively reversed the UVB-induced pathological alterations. These findings were further corroborated by hierarchical clustering heatmaps (Figure 6C,D), which demonstrated that CHE treatment shifted the global gene expression profile of UVB-irradiated cells back towards a pattern resembling the control group.

KEGG pathway analysis (Figure 6E,F) revealed that the overlapping targets were primarily enriched in Environmental Information Processing and Signal Transduction categories. Notably, the PI3K–Akt signaling pathway emerged as a top candidate (3 genes, ~10%), alongside alanine/aspartate metabolism. GO annotation (Figure 6G) further confirmed the involvement of these targets in biological regulation and cellular processes. Collectively, these data identify the PI3K/Akt axis as a pivotal mechanism underlying the anti-photoaging effects of CHE.

2.6. CHE Inhibits the UVB-Induced Hyperactivation of the PI3K/Akt/mTOR Signaling Pathway

To experimentally validate the transcriptomic findings, we examined the phosphorylation status of key kinases in the PI3K/Akt/mTOR axis using Western blot analysis. As shown in Figure 7, UVB irradiation triggered the aberrant activation of this signaling cascade in HSF cells. This was evidenced by the significant upregulation of total PI3K and Akt expression, alongside the hyperphosphorylation of PI3K, Akt, and mTOR (p < 0.001) compared to the control group.

Crucially, CHE intervention effectively reversed this pathological activation in a concentration-dependent manner. Treatment with CHE (50 and 100 μg/mL) significantly downregulated the overexpression of these kinases and potently suppressed the phosphorylation levels of p-PI3K, p-Akt, and p-mTOR (p < 0.05). These proteomic data corroborate the RNA-seq results, confirming that CHE mitigates UVB-induced photodamage by specifically inhibiting the hyperactivated PI3K/Akt/mTOR axis.

3. Discussion

In the present study, we integrated phytochemical profiling, transcriptomic analysis, and multi-dimensional biological assays to elucidate the photoprotective mechanisms of Cardiospermum halicacabum extract (CHE) against UVB-induced senescence in human skin fibroblasts (HSFs). Our primary finding is that CHE effectively mitigates UVB-induced photoaging by suppressing the aberrant hyperactivation of the PI3K/Akt/mTOR signaling axis. This inhibition subsequently restores mitochondrial bioenergetics, reduces intracellular ROS accumulation, and downregulates the senescence markers p53 and p16. To our knowledge, this is the first study to link the photoprotective efficacy of CHE directly to the modulation of the PI3K/Akt/mTOR pathway and mitochondrial homeostasis.

The therapeutic consistency of botanical extracts is often limited by compositional variability [18]. To establish a reproducible quality standard, we characterized the phytochemical profile of CHE using UPLC-MS and HPLC. Our quantitative analysis revealed that CHE is a flavonoid-rich reservoir, containing significant amounts of myricitrin, apigenin, and luteolin [12,19]. These findings are pivotal, as these specific flavonoids are potent inhibitors of the PI3K/Akt/mTOR pathway. For instance, apigenin has been reported to inhibit cell proliferation and induce autophagy by targeting the PI3K/Akt/mTOR axis in UVB-irradiated keratinocytes [20], while luteolin exerts similar protective effects against UVB-induced oxidative stress and DNA damage [21]. The high abundance of these bioactive compounds in CHE provides a solid material basis for its observed anti-photoaging effects.

A critical insight from our study is the identification of the PI3K/Akt/mTOR signaling pathway as the central molecular target of CHE (Figure 8). While the PI3K/Akt axis typically promotes survival, its aberrant hyperactivation under chronic or excessive UVB exposure drives cellular senescence and the accumulation of damaged macromolecules by inhibiting autophagy [16]. Our transcriptomic profiling (RNA-seq) unbiasedly highlighted this pathway as a top candidate, a prediction that was robustly corroborated by Western blot analysis. We observed that CHE treatment effectively reversed the UVB-triggered phosphorylation of PI3K, Akt, and mTOR. This suppression is mechanistically significant because the downregulation of mTOR is intimately linked to the clearance of oxidative damage and the prevention of premature senescence [22,23]. By dampening this overactive signaling node, CHE disrupts the cycle of stress signal amplification, thereby preventing the commitment of HSFs to a senescent state, as evidenced by the profound downregulation of p53 and p16 [24].

The restoration of mitochondrial bioenergetics represents a key downstream functional outcome of CHE-mediated PI3K/Akt/mTOR inhibition. Mitochondria are both the primary source and target of UVB-induced ROS [25]. Our Seahorse metabolic analysis demonstrated that CHE treatment rescued HSFs from UVB-induced bioenergetic collapse, specifically by restoring basal respiration and ATP production while attenuating proton leak. This aligns with the understanding that metabolic regulation is crucial for cell survival under stress [26]. Accordingly, the recovery of mitochondrial function directly contributes to the observed reduction in intracellular ROS levels, breaking the vicious cycle of oxidative stress and mitochondrial impairment [27].

The partial photoprotective efficacy and restricted optimal concentration range (50–100 μg/mL) observed for CHE are characteristic limitations of crude botanical extracts [28]. We postulate that while the enriched flavonoids drive cytoprotection, the complex phytochemical matrix inevitably contains uncharacterized constituents that exert antagonistic effects or non-specific cytotoxicity at elevated doses, thereby capping the maximum efficacy [29]. To this end, future bioassay-guided fractionation is warranted to eliminate these matrix interferences and enrich the active principles [30]. Furthermore, subsequent in vivo studies using animal models are necessary to further evaluate the safety profile and actual therapeutic window of CHE in a physiological environment.

Taken together, our findings position CHE as a superior alternative to conventional synthetic antioxidants, which are often restricted by stability issues and potential toxicity [31]. Unlike simple ROS scavengers, CHE offers a “dual-protection” strategy: it concurrently targets the upstream signaling hub (PI3K/Akt/mTOR) and the downstream energy organelle (mitochondria). This multi-targeted mechanism not only validates the traditional ethnomedicinal use of C. halicacabum [32] but also provides a compelling scientific rationale for its development as a precision-targeted bioactive ingredient. By breaking the vicious cycle of oxidative stress and metabolic failure, CHE represents a promising candidate for next-generation functional cosmeceuticals and therapeutic photoprotection strategies [33,34].

Despite these promising pharmacological findings, several limitations of the present study should be acknowledged. Although our targeted quantitative analysis confirmed the presence of key bioactive flavonoids (myricitrin, luteolin, and apigenin) in CHE, a prominent peak observed at 25 min in the HPLC chromatogram remains unidentified. An extensive screening against a wide array of major phenolic and flavonoid reference standards failed to yield a corresponding match. As is common in the phytochemical profiling of complex botanical matrices, complete characterization is often hindered by the limited availability of commercial standards [35]. Future studies incorporating targeted preparative isolation and Nuclear Magnetic Resonance spectroscopy are warranted to fully elucidate the comprehensive phytochemical profile of this extract.

4. Materials and Methods

4.1. Reagents and Materials

The dried whole plants of Cardiospermum halicacabum L. (Sapindaceae) were collected in September 2023 from Guilin, Guangxi Zhuang Autonomous Region, China. The botanical identification was authenticated by Prof. Xiaohong Yuan (Guangdong Provincial Hospital of Traditional Chinese Medicine), and the specimens were deposited at the Guangdong Academy of Traditional Chinese Medicine. High-performance liquid chromatography (HPLC)-grade reference standards, including myricitrin (DO1031EA14, purity ≥ 98%), luteolin (JB242594, purity ≥ 98%), and apigenin (M29GB150104, purity ≥ 98%), were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). HPLC-grade methanol was acquired from Merck KGaA (Darmstadt, Germany), and phosphoric acid was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). All other chemicals and reagents used were of analytical grade.

4.2. C.halicacabum Extract Preparation

The dried powder of C. halicacabum (1.0 g) was mixed with 30 mL of 70% (v/v) ethanol and subjected to reflux extraction for 45 min. The mixture was filtered, and the residue was washed with 70% ethanol. The combined filtrate was transferred into a volumetric flask, and the volume was made up with 70% ethanol to adjust the concentration. Finally, the solution was filtered through a 0.45 μm microporous membrane to obtain the C. halicacabum extract (CHE) for phytochemical and biological analyses.

Given the inherent phytochemical variability of botanical materials, a single standardized batch of CHE was utilized for all in vitro assays to ensure pharmacological reproducibility. Thus, the exact concentrations of the marker compounds quantified herein serve as the specific phytochemical reference standards responsible for the observed photoprotective efficacy, providing a quality control baseline for future batch-to-batch validations.

4.3. Phytochemical Profiling and Quantitative Analysis

The chemical composition of CHE was comprehensively analyzed using both qualitative UPLC-MS and quantitative HPLC methods.

Qualitative analysis was performed using a Waters ACQUITY UPLC system coupled with a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic separation was achieved on a Waters ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm) maintained at 30 °C. The mobile phase consisted of acetonitrile (solvent A) and 0.1% formic acid in water (solvent B). The gradient elution program was 0–0.5 min, 5% A; 0.5–9.5 min, 5–95% A; 9.5–10 min, 95–99% A; 10–11 min, 99% A. The flow rate was 0.2 mL/min, and the injection volume was 2 μL. Mass spectrometry detection used a heated electrospray ionization (HESI) source in positive and negative modes (spray voltage: 3.5 kV; capillary temp: 350 °C; aux gas: 15 psi; sheath gas: 40/45 psi). Data were acquired in full scan (resolution: 35,000) and DD-MS^2^ (resolution: 17,500) modes using Xcalibur software (version 3.1).

Quantitative analysis of key flavonoids (myricitrin, luteolin, and apigenin) was conducted using an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA). Separation was carried out on an InertSustain AQ-C18 column (250 mm × 4.6 mm, 5 μm) at 30 °C. The mobile phase consisted of methanol (solvent A) and 0.1% phosphoric acid in water (solvent B), following the gradient program listed in Table A1. The flow rate was set at 1.0 mL/min, and the injection volume was 2 μL. Detection was performed at 350 nm. Calibration curves were established using external standards to quantify the target compounds.

4.4. Determination of In Vitro Antioxidant Activity of CHE

4.4.1. Ferric Reducing Antioxidant Power (FRAP) Assay

The ferric reducing potential was determined using a FRAP total antioxidant capacity assay kit (Cat. No. BESBK2607B, BIOESN, Shanghai, China) according to the method described by Hu et al. [36]. Briefly, CHE was diluted in 70% ethanol to a concentration gradient of 0.25, 0.5, 1.0, 2.0, and 2.5 mg/mL, with L-ascorbic acid serving as the positive control. Following the manufacturer’s instructions, 10 μL of the sample or positive control was mixed with 190 μL of the FRAP working solution (comprising acetate buffer, 2,4,6-tripyridyl-s-triazine, and FeCl_3_) in a 96-well plate. After incubation for 20 min at room temperature, the absorbance was measured at 593 nm using a microplate reader. The FRAP value was calculated using the following Equation (1):

$$[eqn]$$

where A_1_ is the absorbance of the sample mixed with the reaction solution, and A_0_ is the absorbance of the blank solvent mixed with the reaction solution. The specific constants (0.8054 and 0.0134) employed in Equation 1 are mathematically derived from the predefined standard calibration curve (y = 1.2416x + 0.0134, where y represents the change in absorbance and x denotes the standard concentration) provided by the manufacturer’s protocol (BIOESN, Shanghai, China). Within this established formula, 0.8054 functions as a derived conversion coefficient to extrapolate the total antioxidant capacity, while 0.0134 corrects for the baseline absorbance.

4.4.2. DPPH Radical Scavenging Assay

The scavenging activity of CHE against the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was assessed using a standard protocol according to the method described by Chen et al. [37]. CHE solutions were prepared in 70% ethanol at concentrations of 0.25, 0.5, 1.0, 2.0, and 2.5 mg/mL, with L-ascorbic acid used as the positive control. A DPPH stock solution (1.5 × 10^−4^ mol/L) was prepared in anhydrous ethanol. For the assay, 80 μL of the sample or positive control was mixed with 120 μL of the DPPH solution in a 96-well plate. The mixture was incubated in the dark for 30 min, and the absorbance was measured at 517 nm. The DPPH radical scavenging activity was calculated according to the following Equation (2):

$$[eqn]$$

where A_0_ represents the absorbance of the negative control (blank solvent with DPPH), and A_1_ denotes the absorbance of the test sample.

4.4.3. ABTS Radical Scavenging Assay

The ABTS radical scavenging capacity was evaluated using an ABTS total antioxidant capacity assay kit (Cat. No. BESBK2623B, BIOESN, Shanghai, China) according to the method described by Dai et al. [38]. CHE samples and L-ascorbic acid controls were prepared in 70% ethanol at the same concentration gradient as described above (0.25–2.5 mg/mL). According to the kit instructions, 80 μL of the sample or control solution was mixed with 120 μL of the ABTS^+^ working solution (with an initial absorbance adjusted to 0.70 ± 0.02 at 734 nm) in a 96-well plate. The absorbance was measured at 734 nm within 10 min. The ABTS radical cation scavenging capacity was determined utilizing the identical Formula (3):

$$[eqn]$$

where A_0_ represents the absorbance of the control (blank solvent with ABTS radical cation), and A_1_ denotes the absorbance of the sample mixture.

4.5. Cell Culture and Viability Assay

Human Skin Fibroblasts (HSFs) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin in a humidified atmosphere of 5% CO_2_ at 37 °C.

Cell viability was determined using the MTT assay. Briefly, HSFs were seeded into 96-well plates at an appropriate density and allowed to adhere for 24 h. The medium was then replaced with fresh medium containing increasing concentrations of CHE (0–6400 μg/mL) for 24 h. Subsequently, 10 μL of MTT solution (5 mg/mL) was added to each well, and the plates were incubated in the dark for 4 h at 37 °C. The supernatant was carefully removed, and 150 μL of DMSO was added to dissolve the formazan crystals. Absorbance was measured at 490 nm using a microplate reader. Cell viability was expressed as a percentage of the control group according to the following Equation (4):

$$[eqn]$$

where A_1_ and A_0_ represent the absorbance of the treated and untreated cells, respectively.

4.6. UVB Irradiation and Experimental Grouping

HSF cells were seeded into 96-well plates at a density of 1 × 10^4^ cells/well and incubated for 24 h. The cells were randomly divided into three groups: the control group (no UVB, no CHE), the UVB model group (UVB irradiation only), and the CHE-treated groups (UVB irradiation + CHE pretreatment).

For the CHE-treated groups, cells were pre-treated with varying concentrations of CHE (25, 50, and 100 μg/mL) for 24 h. Subsequently, the culture medium was removed, and the cells were covered with a thin layer of phosphate-buffered saline (PBS) to prevent drying. UVB irradiation was performed using a UVP UVLMS-38 handheld UV lamp (8 W, 302 nm; Analytik Jena US, Upland, CA, USA). The cells were irradiated at a vertical distance of 15 cm from the light source for 60 s, corresponding to a total energy dose of 48 mJ/cm^2^. This specific irradiation dose was determined through preliminary MTT cell viability assays to optimize the photodamage model, aligning with established protocols [39]. Meanwhile, the control group was sham-irradiated; the cells underwent identical handling procedures but were physically shielded from the UVB source to eliminate potential confounding effects from non-irradiation environmental exposure. After irradiation, the PBS was replaced with fresh medium, and the cells were cultured for an additional 24 h. Finally, cell viability was measured using the MTT assay as described in Section 4.5.

4.7. Measurement of Intracellular ROS Accumulation

Intracellular ROS levels were quantified using the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe. Briefly, HSF cells were seeded in 6-well plates at a density of 3 × 10^5^ cells/well. Consistent with the grouping described in Section 2.6, cells were pre-treated with CHE (25, 50, and 100 μg/mL) for 24 h prior to UVB irradiation (48 mJ/cm^2^).

Following the treatment, cells were washed with PBS and incubated with 10 μM DCFH-DA in serum-free medium for 30 min at 37 °C in the dark. Excess dye was removed by washing the cells three times with PBS. The cells were then harvested by trypsinization, resuspended in PBS, and immediately analyzed using a flow cytometer (excitation: 488 nm; emission: 525 nm). The mean fluorescence intensity (MFI) was calculated to reflect the intracellular ROS levels.

4.8. Mitochondrial Respiration Analysis (Seahorse Assay)

Mitochondrial oxygen consumption rates (OCR) were quantified using the Agilent Seahorse XFe24 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA; RRID: SCR_019539). Briefly, HSF cells were seeded into XF24 cell culture microplates and subjected to CHE pretreatment and UVB irradiation as detailed in Section 2.6. Mitochondrial function was assessed using the Agilent Seahorse XF Cell Mito Stress Test Kit according to the manufacturer’s instructions. Prior to the assay, the sensor cartridges were hydrated overnight in XF Calibrant at 37 °C in a non-CO_2_ incubator. On the day of analysis, the culture medium was replaced with XF base medium (pH 7.4) supplemented with 10 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate, and the cells were equilibrated for 1 h in a non-CO_2_ incubator. During the assay, metabolic modulators provided in the kit were sequentially injected to achieve final well concentrations of 1 μM oligomycin, 1 μM FCCP, and 0.5 μM rotenone/antimycin A. Post-assay, cells were trypsinized and counted using a Countstar automated cell counter to normalize the OCR data. Data acquisition and processing were performed using the Wave Controller software (version 2.6.4; RRID: SCR_024491).

4.9. Cell Cycle Analysis

Cell cycle distribution was determined using Propidium Iodide (PI) staining flow cytometry. Following CHE pretreatment and UVB irradiation as described in Section 2.6, HSF cells were harvested by trypsinization and washed with ice-cold PBS. The cells were fixed in 70% (v/v) ethanol at 4 °C overnight. After fixation, the cells were washed twice with PBS to remove ethanol and resuspended in a PI/RNase staining solution. The samples were incubated in the dark at room temperature for 30 min. Finally, DNA content was analyzed using a NovoCyte Quanteon flow cytometer (Agilent Technologies, Santa Clara, CA, USA). The percentage of cells in the G0/G1, S, and G2/M phases was calculated using the accompanying analysis software.

4.10. Transcriptome Sequencing and Bioinformatics Analysis

Total RNA was extracted from HSF cells in the Control, UVB-irradiated, and CHE-treated groups (three biological replicates per group) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) followed by purification with the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA concentration and integrity were verified prior to library construction. cDNA libraries were generated and sequenced on an Illumina platform by Metware Biotechnology Co., Ltd. (Wuhan, China). Raw sequencing data were filtered and aligned to the reference genome. Differentially expressed genes (DEGs) were identified using the DESeq2 R package (version 1.42.0), with a significance threshold of p < 0.05 and |log_2_Fold Change| > 1.

4.11. Western Blot Analysis

Total protein was extracted from HSF cells using RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktails. The protein concentration was determined using the BCA protein assay kit. Equal amounts of protein (30 μg) were separated by SDS-PAGE and transferred onto methanol-activated PVDF membranes. The membranes were blocked with 5% skim milk in TBST for 1 h at room temperature and then incubated overnight at 4 °C with specific primary antibodies from Proteintech (Wuhan, China): PI3K (60225-1-Ig), p-PI3K (28856-1-AP), Akt (10176-2-AP), p-Akt (66444-1-Ig), mTOR (66888-1-Ig), p-mTOR (67778-1-Ig), p53 (10442-1-AP), p-p53 (28961-1-AP), p16 (10883-1-AP), and β-actin (66009-1-Ig). All antibodies were diluted 1:1000. Following three washes with TBST, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) kit and imaged using a chemiluminescent imaging system. Band intensities were quantified via densitometric analysis using ImageJ software (version 1.53c; NIH, Bethesda, MD, USA).

4.12. Statistical Analysis

All quantitative data are expressed as mean ± standard deviation (SD) derived from at least three independent experiments. Statistical analyses were performed using SPSS 26.0 software (IBM, Armonk, NY, USA) and GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Differences between two groups were analyzed using Student’s t-test. For comparisons among three or more groups, a one-way analysis of variance (ANOVA) was performed, followed by Tukey’s post hoc test. A p-value < 0.05 was considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001).

5. Conclusions

This study establishes Cardiospermum halicacabum extract (CHE) as a potent anti-photoaging agent, chemically standardized by its high content of myricitrin, apigenin, and luteolin. Mechanistically, we demonstrate that CHE exerts its cytoprotective effects by specifically inhibiting the UVB-triggered hyperactivation of the PI3K/Akt/mTOR axis. This targeted intervention effectively restores mitochondrial bioenergetics and arrests cellular senescence, providing a scientific validation for the traditional use of C. halicacabum. While our in vitro findings highlight CHE as a superior, multi-target alternative to synthetic antioxidants, future translational research should prioritize in vivo validation and the development of transdermal delivery systems to maximize its potential in next-generation functional cosmeceuticals.