**2. Results**

### *2.1. FCP Promote Cell Proliferation and Inhibit Cisplatin-Induced Cytotoxicity*

A WST-1-based colorimetric cell proliferation assay was used evaluate the ability of FCP to facilitate cell proliferation. Treatment of TECs with FCP for 24 h significantly enhanced cell proliferation at concentrations of 0.01, 0.05, 0.08, 0.1 and 0.15%, by 19.6% (*p* < 0.01), 23.7% (*p* < 0.001), 27.1% (*p* < 0.001), 19.3% (*p* < 0.01), and 11.2% (*p* < 0.05), respectively, compared with the control (Figure 1). At 48 h, the rates of cell proliferation in the FCP-treated group versus the control at concentrations of 0.01%, 0.05%, 0.08%, 0.1%, 0.15%, and 0.2% were greatly increased by 30.9% (*p* < 0.001), 44.8% (*p* < 0.001), 56% (*p* < 0.001), 38.6% (*p* < 0.001), 21.7% (*p* < 0.01), and 12.8% (*p* < 0.01), respectively, compared with the control (Figure 1).

Cellular cytotoxicity and morphology were assessed to examine the level of cisplatininduced cell injury. Here, the WST-1-based colorimetric cell viability assay after treatment with cisplatin for 24 h at concentrations of 5, 10, and 20 μM revealed a significant decrease in cell number by 26.7% (*p* < 0.01), 36.9% (*p* < 0.01), and 57.9% (*p* < 0.001), respectively, relative to the control (Figure 2). Cisplatin treatment for 48 h at concentrations of 5, 10,

and 20 μM strongly reduced cell viability by 61.3% (*p* < 0.001), 87.9% (*p* < 0.001), and 100% (*p* < 0.001), respectively, compared with the control (Figure 2). These cytotoxicity results were also concurrent with the morphological changes observed by phase contrast microscopy (Figure 2), revealing dose- and time-dependent cytotoxic effects of cisplatin in TECs.

**Figure 1.** Stimulatory effects of FCP on cell proliferation. Proliferation of TECs was measured by phase contrast microscopy (**A**) and cell viability assay (**B**) as described in Materials and Methods. The treatment of TECs with FCP for 24 and 48 h significantly enhanced cell proliferation. Results are presented as the means ± SD of three independent experiments. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. the control at 24 h; ## *p* < 0.01, ### *p* < 0.001 vs. the control at 48 h. Scale bar = 50 μm.

Subsequently, the protective effect of FCP was investigated on cisplatin-induced cytotoxicity in TECs by WST-1 using the phase contrast microscopic assays. As shown in Figure 3, exposure of TECs to cisplatin at concentrations of 5, 7.5, 10 and 15 μM for 24 h led to a reduced cell viability, by 22.5% (*p* < 0.01), 30.3% (*p* < 0.001), 43.1% (*p* < 0.001), and 58.7% (*p* < 0.001), respectively, compared with the control. However, 0.08% FCP pretreatment prior to cisplatin treatment (5, 7.5, 10, and 15 μM for 24 h) resulted in the enhancement of cellular viability, by 41.1% (*p* < 0.001), 42.5% (*p* < 0.01), 36.7% (*p* < 0.01), and 24.04% (*p* < 0.001), respectively, compared with the cisplatin alone treatment group. This result, therefore, indicates the protective role of FCP against cisplatin-induced TEC damage (Figure 3).

Furthermore, molecular mechanisms underlying the protective effect of FCP on TECs injured by cisplatin were explored by analyzing the expression of apoptosis- and cell cyclerelated proteins. Cisplatin treatment significantly reduced the expression of anti-apoptotic molecules, Bcl-2 and Bcl-xL, by 24.4% (*p* < 0.001) and 21.2% (*p* < 0.001), respectively, and enhanced the expression of pro-apoptotic molecules, Bax, Bad and cytochrome-c, by 40.1% (*p* < 0.001), 20.8% (*p* < 0.001), and 40.5% (*p* < 0.01), respectively (Figure 4A), whereas it significantly suppressed the expression of the key cell cycle regulatory molecules, cyclin D1 and CDK1 proteins by 52.1% (*p* < 0.001) and 35.5% (*p* < 0.001), respectively, compared with the untreated control (Figure 4B). Notably, all these cisplatin-induced alterations in the expression of apoptosis- and cell cycle-related proteins almost returned to their normal levels after 0.08% FCP pretreatment for 24 h. The cisplatin-induced downregulated expression of Bcl-2, Bcl-xL, cyclin D1, and CDK1 proteins increased due to FCP by 26% (*p* < 0.001),

22.6% (*p* < 0.01), 29.9% (*p* < 0.05), and 42.8% (*p* < 0.001), respectively, (Figure 4A,B), whereas the cisplatin-induced upregulated expression of Bax, Bad and cytochrome-c was reversed following exposure to FCP by 58% (*p* < 0.001), 39.3% (*p* < 0.001), and 29% (*p* < 0.001), respectively, compared with the cisplatin alone treatment group (Figure 4A).

**Figure 2.** Cytotoxic effects of cisplatin on TECs were measured by phase contrast microscopy (**A**) and cell cytotoxicity assay (**B**) as described in Materials and Methods. The treatment of TECs with 5, 10, and 20 μM cisplatin for 24 and 48 h significantly attenuated cell viability. Results are presented as the means ± SD of three independent experiments. \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. the control at 24 h; ### *p* < 0.001 vs. the control at 48 h. Scale bar = 50 μm.

**Figure 3.** Protective effect of FCP on cisplatin-induced cytotoxicity in TECs. Cell viability was measured by phase contrast microscopy (**A**) and WST-1 assay (**B**) in TECs as described in Materials and Methods. The decreased cell number induced by cisplatin treatment (5, 7.5, 10, and 15 μM) was significantly restored by treatment with 0.08% FCP for 24 h. Results are presented as the means ± SD of three independent experiments. \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. the control. ## *p* < 0.01, ### *p* < 0.001 vs. the cisplatin alone-treated group. Scale bar = 50 μm.

**Figure 4.** Western blot analysis on the inhibitory effects of FCP on cisplatin-induced altered expression of apoptosis- and proliferation-related proteins in TECs. FCP pretreatment in cisplatin alone-treated TECs significantly enhanced the levels of Bcl-2 and Bcl-xL, reduced the levels of Bad, Bax, and cytochrome-c (**A**), and elevated the levels of cyclin D1 and CDK1 compar with the cisplatin alonetreated group (**B**). Results are presented as the means ± SD of three independent experiments. \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. the control. # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001 vs. the cisplatin alonetreated group.

### *2.2. FCP Attenuate Cisplatin-Induced ROS Generation*

To measure changes in the cellular levels of ROS in response to cisplatin with or without FCP pretreatment, TECs were pretreated with 0.08% FCP for 24 h followed by cisplatin exposure at 10 μM for 24 h (Figure 5A,B). Treatment with cisplatin significantly increased the ROS level as detected by fluorescence microscopy using the oxidant-sensing fluorescent probe <sup>2</sup>,7-dichlorodihydrofluorescein diacetate (DCFH-DA) by 34.4% (*p* < 0.05) compared with the control (Figure 5A,B).

Pretreatment with FCP showed a significant decrease in the cisplatin-induced ROS release by 72.1% (*p* < 0.01) compared with the cisplatin alone-treated group, indicating its restorative effect on the endogenous antioxidant defense mechanism impaired by cisplatin (Figure 5A,B). Furthermore, treatment with N-acetyl cysteine (NAC), a common ROS scavenger, at a concentration of 5 mM for 2 h, significantly reduced the cisplatin-enhanced ROS level in TECs, by 69.2% (*p* < 0.001) compared with the cisplatin alone-treated group (Figure 5A,B). These findings also indicate that the inhibitory action of FCP on cisplatininduced cytotoxicity in TECs is attributed to its antioxidant effect.

**Figure 5.** Inhibitory effects of FCP and NAC on cisplatin-induced ROS generation in TECs. Intracellular ROS levels were determined via fluorescence microscopy (**A**) and spectroscopy (**B**) using DCFH-DA in TECs. The increased ROS level induced by 10 μM cisplatin treatment was significantly attenuated by pretreatment with 0.08% FCP for 24 h and NAC for 2 h. Quantification of staining intensity was measured by ImageJ software. Results are presented as the means ± SD of three independent experiments. \* *p* < 0.05, \*\*\* *p* < 0.001 vs. the control. ## *p* < 0.01, ### *p* < 0.001 vs. the cisplatin alone-treated group. Scale bar = 50 μm.

### *2.3. FCP Alleviate Cisplatin-Induced TEC Cytotoxicity through Suppression of MAPK Pathway*

MAPK cascades are key signaling pathways that regulate various cellular processes, including stress and inflammatory responses, as well as cell proliferation, differentiation, apoptosis, and motility under both normal and pathological conditions [29,30]. Since cisplatin-induced cell death has been shown to be dependent on the MAPK pathways in several cell types [31], we first tested if cisplatin could activate this pathway in TECs. The results of this study revealed an increase in the expression of p-p38 MAPK, p-JNK, and p-ERK by 57.9% (*p* < 0.001), 16.8% (*p* < 0.001), and 21.8% (*p* < 0.001), in the cisplatin alone-treated group, respectively, than in the control, whereas the expression of total p38 MAPK, JNK, and ERK levels remained unaltered, indicating the activation of p38 MAPK, JNK, and ERK pathways after cisplatin treatment in TECs (Figure 6). However, these elevated levels of p-p38 MAPK, p-JNK and p-ERK were attenuated by the pretreatment with 0.08% FCP for 24 h, by 37.3% (*p* < 0.01), 15.3% (*p* < 0.001), and 58.5% (*p* < 0.001), respectively, compared with the cisplatin alone-treated group (Figure 6).

To further explore the role of MAPK pathways in cisplatin-induced cell cytotoxicity, the cells were treated with a selective p38 MAPK inhibitor (SB203580), JNK inhibitor (SP600125), or ERK inhibitor (U0126). Analysis of the cell viability by WST-1 assay after TECs were treated with 0.08% FCP, 10 μM SB203580, SP600125, and U0126 for 24 h, followed by treatment with or without 10 μM cisplatin for 24 h, showed that FCP pretreatment caused a significant reduction of cisplatin-induced cytotoxicity, similar to all MAPK inhibitor pretreatments, whereas SB203580, SP600125 or U0126 alone did not affect cell viability (Figure 7).

Confirmation of the effect of FCP treatment on the cisplatin-induced expression of MAPK was performed using western blot analysis after TECs were treated with 0.08% FCP and 10 μM SB203580, SP600125, and U0126 for 24 h, followed by treatment with or without 10 μM cisplatin for 24 h. Notably, we observed that FCP pretreatment potently repressed

the cisplatin-induced upregulated expression of p-p38 MAPK, p-JNK, and p-ERK, as was the case with MAPK inhibitor pretreatment (Figure 8A–C).

**Figure 6.** Inhibitory effects of FCP on cisplatin-induced activation of p38 MAPK, JNK, and ERK signaling pathway. The expression of p38 MAPK, JNK, and ERK was increased in the cisplatin alone-treated TECs, as assessed by Western blot analysis (**A**). The pretreatment of TECs with FCP blocked the cisplatin-induced phosphorylation of p38 MAPK, JNK, and ERK. Bar graphs depict relative densitometry quantitation of each protein normalized to β-actin (**B**). Results are presented as the means ± SD of three independent experiments. \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. the control. ## *p* < 0.01, ### *p* < 0.001 vs. the cisplatin alone-treated group.

**Figure 7.** FCP promotes TECs proliferation via activation of p38 MAPK (**A**), JNK (**B**), and ERK (**C**) signaling pathways. The treatment of TECs with FCP for 24 h significantly enhanced cell proliferation. The cisplatin-induced cell cytotoxicity was recovered by treatment with FCP, SB203580, SP600125, and U0126 in TECs. Results are presented as the means ± SD of three independent experiments. \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. the control at 24 h; # *p* < 0.05, ## *p* < 0.01, vs. the control at 48 h.

Together, these results indicate that FCP acts as a potent inhibitor of MAPK pathways, which exerts a protective effect against cisplatin-induced cytotoxicity via suppression of p38 MAPK, JNK, and ERK pathway in TECs.

### *2.4. FCP Prevent Cisplatin-Induced ROS Generation by Inhibition of MAPK Signaling*

To investigate whether p38 MAPK, JNK and ERK activation is associated with cisplatininduced oxidative cell injury in TECs, we examined the effect of p38 MAPK, JNK and ERK inhibition on the cisplatin-induced ROS production. We also assessed the protective effect of FCP on cisplatin-induced ROS release in TECs using the DCFH-DA assay. As shown in Figure 9A–C, the exposure of TECs to 10 μM cisplatin for 24 h caused an increase in ROS level, by 29.5% (*p* < 0.01), 34% (*p* < 0.001) and 33.8% (*p* < 0.001) *versus* the control. However, the enhanced level of ROS induced by cisplatin treatment was significantly

declined by the treatment with 10 μM SB203580, 10 μM SP600125 or 10 μM U0126 for 24 h by 25.2% (*p* < 0.01), 46.3% (*p* < 0.01), and 29.6% (*p* < 0.05), respectively, and with 0.08% FCP for 24 h by 37.8% (*p* < 0.01), 79% (*p* < 0.001) and 50.3% (*p* < 0.05), respectively (Figure 9A–C).

**Figure 8.** Inhibitory effects of FCP, SB203580 (**A**), SP600125 (**B**), and U0126 (**C**) on cisplatin-induced activation of p38 MAPK, JNK, and ERK signaling pathway in TECs. The expression of p-p38 MAPK, p-JNK, and p-ERK was increased in cisplatin-treated TECs. The pretreatment of TECs with FCP, SB203580 (**A**), SP600125 (**B**), and U0126 (**C**) blocked the cisplatin-induced phosphorylation of p38 MAPK, JNK, and ERK. Results are presented as the means ± SD of three independent experiments. \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. the control. ### *p* < 0.001 vs. the cisplatin alone-treated group.

As shown in Figure 10, the treatment of TECs with 10 μM cisplatin for 24 h resulted in the marked upregulation of p-p38 MAPK, p-JNK, and p-ERK expression compared with the control group by 72.1% (*p* < 0.001), 28.8% (*p* < 0.05), and 56.6% (*p* < 0.001), respectively, while the amount of total p38 MAPK, p-JNK, and p-ERK levels were unaltered. To decipher the role of p38 MAPK, JNK, and ERK in cisplatin-induced oxidative cellular injury in TECs, we investigated the effect of NAC on the cisplatin-induced expression of p-p38 MAPK, p-JNK, and p-ERK in TECs. Pretreatment of cells with FCP for 24 h or NAC for 2 h prior to cisplatin treatment completely abolished the cisplatin-induced phosphorylation of p38 MAPK, JNK, and ERK (Figure 10). These findings indicate that p38 MAPK, JNK and ERK activation is important in cisplatin-induced cellular oxidative stress, and FCP exhibits a strong protective effect against cisplatin-induced oxidative cell injury via the suppression of p38 MAPK, JNK, and ERK pathways in TECs.

**Figure 9.** Inhibitory effects of FCP on cisplatin-induced ROS generation in TECs via activation of p38 MAPK, JNK and ERK signaling pathway. Intracellular ROS levels were determined via fluorescence microscopy (a) and spectroscopy (b). The increased ROS production induced by cisplatin returned to the control level by treatment with FCP, SB203580 (**A**), SP600125 (**B**) and U0126 (**C**) in TECs. Results are presented as the means ± SD of three independent experiments. \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. the control. # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001 vs. the cisplatin alone-treated group. Scale bar = 50 μm.

**Figure 10.** Inhibitory effects of FCP and NAC on cisplatin-induced activation of p38 MAPK, JNK, and ERK signaling pathways in TECs. The expression of p38 MAPK, JNK, and ERK was increased in cisplatin-treated TECs, as assessed by Western blot analysis (**A**). The pretreatment of TECs with FCP or NAC blocked the cisplatin-induced phosphorylation of p38 MAPK, JNK, and ERK. Bar graphs depict relative densitometry quantitation of each protein normalized to β-actin (**B**). Results are presented as the means ± SD of three independent experiments. \* *p* < 0.01, \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. the control. # *p* < 0.05, ### *p* < 0.001 vs. the cisplatin alone-treated group.
