Triclosan affects steroidogenesis in mouse primary astrocytes in vitro with engagement of Sirtuin 1 and 3

Triclosan (TCS) is a widely used antimicrobial, antifungal, and antiviral agent. To date, it has been reported that TCS can enter the human body and disrupt hormonal homeostasis. Therefore, the aim of our paper was to evaluate the impact of TCS on astrocytes, i.e. a crucial population of cells responsible for steroid hormone production. Our data showed that, in mouse primary astrocyte cultures, TCS can act as an endocrine disrupting chemical through destabilization of the production or secretion of progesterone (P 4 ), testosterone (T), and estradiol (E 2 ). TCS affects the mRNA expression of enzymes involved in neurosteroidogenesis, such as Cyp17a1 , 17 β -Hsd, and Cyp19a1 . Our data showed that a partial PPAR γ agonist (honokiol) prevented changes in Cyp17a1 mRNA expression caused by TCS. Similarly, honokiol inhibited TCS-stimulated P 4 release. However, rosiglita-zone (classic PPAR γ agonist) or GW9662 (PPAR γ antagonist) had a much stronger effect. Therefore, we believe that the changes observed in the P 4 , T, and E 2 levels are a result of dysregulation of the activity of the afore-mentioned enzymes, whose expression can be affected by TCS through a Ppar γ -dependent pathway. TCS was found to decrease the aryl hydrocarbon receptor (AhR) and Sirtuin 3 protein levels, which may be the result of the activation of the these proteins. Since our study showed dysregulation of the production or secretion of neurosteroids in astrocytes, it can be concluded that TCS reaching the brain may contribute to the development of neurodegenerative diseases in which an abnormal amount of neurosteroids is observed.


Introduction
Triclosan (TCS, CAS no.3380-34-5, 5-chloro-2-(2,4-dichlorophenoxy) phenol) is a widely used antimicrobial, antifungal, and antiviral agent [1].This compound can be found in a number of medical devices, personal care products, household disinfectants, textiles, paper, or even garden furniture [2].Since TCS is not closely bound to the structure of the material, it is easily released from the products containing this compound and commonly enters the environment through wastewater [3].As a contaminant, TCS has been detected in surface and drinking waters, with which it can enter the human body [4,5].This compound can also be absorbed through human skin and oral mucosa and is found in various human tissues and fluids, such as milk and blood of nursing mothers [6,7].Moreover, TCS was detected in urine samples with concentrations ranging from 0.08 to 0.71 µM [8], which suggests its higher amount in the human body.
Currently, it is generally accepted that TCS is an endocrinedisrupting compound (EDC).By its estrogenic activity, TCS affects the reproductive system in both males and females in in vitro and in vivo conditions as well as in a wide range of species [reviewed in [9]].A number of EDCs have well-documented properties leading to impairment of nervous system functioning [reviewed in [10]].Therefore, TCS has been studied in neuronal cultures and in the nervous system of animals in vivo.Primarily, in the nervous system, TCS can affect neuronal cells through increased production of reactive oxygen species (ROS) [reviewed in [11]].However, several papers describe that, in µM concentrations, TCS initiates apoptosis in primary mouse neurons via aryl hydrocarbon receptor (AhR)-dependent pathways and, at least partially, through peroxisome proliferator-activated receptor gamma (Pparγ) [12][13][14].Both AhR and PPARγ can affect brain neurosteroid production and secretion [15,16].Moreover, these receptors are in crosstalk during which agonists and/or antagonists of one receptor can affect each other [17].To date, it has been described that 10 µM TCS produces a decrease in progesterone (P 4 ), testosterone (T), and estradiol (E 2 ) production in mouse primary neurons in vitro via an AhR-dependent way [18].Unfortunately, the role of PPARγ in this process is unclear.
Honokiol is a natural compound which was isolated from Magnolia grandiflora and Magnolia dealbata [19].To date, honokiol has been identified as a new partial non-adaptogenic PPARγ agonist [20].Moreover, it is currently being tested for use as an antioxidant, antidiabetic, anticancer, anti-inflammatory, and neuroprotective agent in multiple in vitro and in vivo studies [21].Honokiol easily crosses the blood-brain barrier (BBB) and affects mouse astrocytes [22].Moreover, it prevents chronic cerebral hypoperfusion-induced astrocyte polarization to alleviate neurotoxicity by targeting the Sirtuin 3 (Sirt3)-Stat3 pathway [23].This may be possible due to Sirt1 and Sirt3 being activated by Pparγ [24,25], but this requires further studies, especially in the context of TCS.
Despite the key role of astrocytes in the production of neurosteroids and the maintenance of neurons, no studies have been performed on the effect of TCS on astrocytes and their functioning.Therefore, the aim of the present study was to determine the impact of TCS on primary astrocyte steroid hormone production and the involvement of Sirt1 and Sirt3 in this process.

Primary astrocyte culture and treatment
The experiments were performed on primary mouse astrocytes isolated from fetuses of pregnant female Swiss mice.All procedures and necessary permits from bioethics committees have been earlier described in detail in Szychowski et al. [26].The astrocytes were cultivated in DMEM/F12 medium without phenol red supplemented with 10 % CH-FBS, 50 U/mL of penicillin, and 0.05 mg/mL of streptomycin.CH-FBS is dedicated to testing substances suspected of having endocrine-disrupting properties.The astrocytes were seeded at a density of 5 × 10 5 per well in a 96-well plate for the lactate dehydrogenase (LDH) measurement and 50 × 10 5 per well in a 12-well plate for the study of mRNA expression and steroid hormone production and/or release.The total RNA (for RT-PCR) concentrations were determined spectrophotometrically in triplicate using ND/1000 UV/Vis (Thermo Fisher NanoDrop, USA).For the LDH release assay, the astrocytes were exposed to a wide range (1 nM to 100 µM) of TCS concentrations.For determination of gene and protein expression as well as hormone production and secretion, the astrocytes were exposed to 1 µM TCS, 10 µM honokiol (HON), 1 µM rosiglitazone (ROSI), and 1 µM GW9662.In the co-treatment experiments, the cells were exposed to tool compounds (HON, ROSI or GW9662) 30 min before TCS treatment.The final DMSO concentration in the culture medium was always equal to 0.01 %.

LDH measurement
The toxicity of TCS in primary astrocytes cells was studied using the LDH release assay as described previously [27].Briefly, after 24-h exposure, the culture medium was transferred to a new 96-well plate.The reaction solution was added and incubated for 30 min at RT in the dark.The absorbance was measured on the microplate reader (FilterMax F5, Molecular Devices, Corp., Sunnyvale, CA, USA) at 490 nm.

ELISA measurement for steroids
The level of steroid hormones (P 4, T, and E 2 ) was determined after 24 h using the enzyme-linked immunosorbent assay (ELISA) method according to the producers' manuals (DRG MedTek).The absorbance was measured at 450 nm and the obtained value was inversely proportional to the level of P 4 , T, or E 2 .The final results were standardized to the amount of total protein content in each sample (measured with the Bradford method).

ELISA measurement for proteins
The levels of the Pparγ, AhR, Sirt1, and Sirt3 proteins were determined after 24 h using ELISA, providing specific detections of these proteins, according to the producers' manuals (Elabscience Biotechnology).The absorbance was measured at 450 nm using a microplate reader (FilterMax F5).This value was proportional to the amount of Pparγ, AhR, Sirt1, or Sirt3.The final results were first standardized to the total protein content in each sample (with the Bradford method).

Real-time PCR
Total RNA was extracted from the astrocytes using the Universal RNA Purification Kit according to the manufacturer's protocol.The reverse transcription reaction was performed according to the manufacturer's protocol (ThermoFisher) at a final volume of 20 μL with 500 ng of RNA (as a cDNA template).Real-time PCR was performed using the Fast Probe qPCR Master Mix (2x) kit with TaqMan probes for specific genes in a total volume of 20 μL containing 1 μL of the cDNA.
The standard qPCR procedure was performed as follows: 2 min at 50 • C and 10 min at 95 • C followed by 40 cycles of 15 s at 95 • C and 1 min at 60 • C. Gapdh was used as a reference gene.The RefFinder web-based comprehensive tool was used to evaluate the reference gene expression.

Statistical analysis
The data are presented as means ± SD of three independent experiments.Each treatment was repeated at least 3 times.The experimental data were analyzed with one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test with * p < 0.05, ** p < 0.01, and *** p < 0.001, compared to the control cells.

TCS toxicity and proliferation and apoptotic markers
After the 24-h exposure of the primary mouse astrocytes to the increasing (1 nM to 100 µM) TCS concentrations, the LDH release increased slightly but statistically significantly by 6.7, 8.4, and 8.2 % at the 10, 50, and 100 µM concentrations, respectively, compared to the control (Fig. 1A).In this time interval, the 1 and 10 µM TCS concentrations caused a 25.18 and 12.22 % decrease in the Ki67 mRNA expression, respectively, compared to the control (Fig. 1B).In turn, the studied concentrations of TCS did not affect the p53 mRNA expression (Fig. 1C).

Gene expression
After the 24-h exposure to TCS and HON, both studied compounds decreased the levels of Nf-κb mRNA by 54.94 and 33.32 % respectively, compared to the control (Fig. 2 A).The TCS and HON co-treated cells exhibited an 11.12 % increase in Nf-κb mRNA, compared to the TCS alone-treated control cells.In these experiments, TCS alone did not significantly affect the Pparγ mRNA expression, while HON produced a 20.42 % decrease in Pparγ mRNA expression, compared to the control (Fig. 2 B).However, a 52.42 % increase in Pparγ mRNA expression was observed in the TCS and HON co-treated cells, compared to the control.Interestingly, TCS alone increased the AhR mRNA expression by 29.09 %, compared to the control, while a 25.67 and 87.85 % decrease in the AhR mRNA expression was observed in the HON alone treatment and the TCS with HON co-treated group, respectively (Fig. 2 C).
In addition to the transcription factors described above, TCS also affected the mRNA expression of enzymes involved in steroidogenesis.TCS increased the Cyp17a1 mRNA expression by 666.91 %, compared to the control (Fig. 2 D).HON alone decreased the Cyp17a1 mRNA expression by 65.81 %, compared to the control.Interestingly, no changes were observed in the TCS and HON co-treated group, compared to the control.In turn an 18.04, 26.10, and 30.24 % decrease in the 17β-Hsd mRNA expression was observed in all the studied groups: TCS, HON, and TCS with HON, respectively, compared to the control (Fig. 2 E).Moreover, the astrocyte co-treatment with TCS and HON decreased the 17β-Hsd mRNA expression by 12.20 %, compared to the TCS alone treatment.Finally, TCS and HON alone decreased the Cyp19a1 mRNA expression by 67.93 and 63.08 %, respectively, compared to the control (Fig. 2 F).In turn, an 852.07 % increase in the Cyp19a1 mRNA expression was observed in the group co-treated with TCS and HON, compared to the control.

Steroid hormone production and release
After the 24-h exposure of the astrocytes to the studied compounds, TCS alone did not affect the P 4 production, but HON increased the P 4 production by 1.34 ng/mL, compared to the control (Fig. 3 A).The astrocyte co-treatment with TCS and HON did not affect the P 4 production.However, TCS increased the P 4 secretion to the culture medium by 2.46 ng/mL (Fig. 3 B).Similarly, HON alone increased the P 4 secretion to the culture medium by 1.82 ng/mL.The other tool compounds, i. e. ROSI (Pparγ agonist) and GW9662 (Pparγ antagonist), did not affect the P 4 secretion.Interestingly, these data suggest that TCS interferes with the HON, ROSI, and GW9662 mechanism of action.
In our experiments, TCS decreased the T production in the astrocytes by 1.53 ng/mL compared to the control.Similarly, in all the studied groups: HON with TCS, ROIS, ROSI with TCS, GW9662, and GW9662 with TCS, we observed a 1.28, 1.25, 1.63, 2.34, and 2.59 ng/mL decrease in the T production, respectively, compared to the control (Fig. 3 C).In the studied cells, TCS, HON alone, and TCS together with HON increased the T secretion to the culture medium by 1.61, 0.90, and 1.24 ng/mL, respectively, compared to the control.Interestingly, ROSI decreased the T secretion by 1.74 ng/mL, while no changes were observed in the group co-treated with TCS and ROSI, compared to the control.
After the 24-h exposure, TCS decreased the E 2 production in the astrocytes by 102.02 pg/mL (Fig. 3 E).Similarly, a 122.32 and 160.72 pg/mL decrease in the E 2 production was observed in the groups treated with HON with TCS and ROSI, respectively, compared to the control.In our experiments, we did not detect E 2 in the astrocyte culture medium.

Protein expression
In our experiments, no changes in the Pparγ protein level were observed in any of the studied experimental group, compared to the control (Fig. 4 A).
After the 24-h exposure, TCS decreased the AhR protein level in the astrocyte culture by 1.16 ng/mL, compared to the control (Fig. 4 B).The tool compounds alone (HON, ROSI, and GW9662) did not significantly change the AhR protein expression.In turn, the AhR protein level in the astrocytes co-treated with TCS with HON, TCS with ROSI, and TCS with GW9662 decreased by 1.08, 1.54, and 1.86 ng/mL, respectively, compared to the control.
In our experiments, HON increase the Sirt1 protein expression by 5.96 ng/mL, compared to the control (Fig. 4 C).Interestingly, in the astrocytes co-treated with HON and TCS, a 3.96 ng/mL decrease in the Sirt1 level was observed, compared to the control.In the groups cotreated with ROSI with TCS and GW9662 alone and in the cells cotreated with GW9662 with TCS, the Sirt1 protein expression increased by 4.14, 5.34, and 4.33 ng/mL respectively, compared to the control.
Lastly, in our experiments, TCS, HON, ROSI, and GW9662 alone decreased the Sirt3 protein expression by 67.16, 55.37, 54.14, and 57.23 pg/mL, respectively, compared to the control (Fig. 4 D).Similarly, the Sirt3 protein expression decreased by 37.82 and 63.32 pg/mL in the groups co-treated with HON and TCS as well as ROSI and TSC, respectively, compared to the control.

Discussion
Our data show relatively low toxicity of TCS to mouse primary astrocytes in vitro.The LDH release increased slightly but statistically significantly only at the 10, 50, and 100 µM concentrations.Moreover, 1 and 10 µM TCS decreased the Ki67 mRNA expression (marker of proliferation) but did not affect the p53 mRNA expression (marker of apoptosis).Therefore, it can be concluded that TCS is not strongly toxic to astrocyte cultures.To date, significant toxicity of TCS applied in the range of 0.1-20 µM has been described in mouse primary neocortical mouse neurons [28], the mouse immortalized microglial (BV-2) cell line [29], the immortalized hippocampal neurons (HT-22) cell line [30], rat neural stem cells (NSCs) [31], and the rat adrenal medulla (PC12) cell line [32].The higher resistance of astrocytes compared to neurons may be a result of higher levels of endogenous antioxidants and more effective metabolic pathways [33].These properties make astrocytes more resistant to oxidative stress, which is the one of the main mechanisms of TCS action [11].
For our further studies, we chose 1 µM TCS, i.e. the first concentrations that did not significantly increase the LDH release and was close to TCS concentrations detected in the human body.Furthermore, the lack of LDH release is an important indicator of cell membrane integrity.Hence, we are sure that the observed hormone secretion was not a case of efflux from damaged cells.Our data show a TCS-induced increase in the P 4 and T secretion to the medium and a decrease in the T and E 2 production in the astrocyte culture.However, in our previous studies on mouse primary neurons, 10 µM TCS decreased the P 4 , T, and E 2 production through an AhR-dependent pathway [18].Similarly, Kumar et al. (2008Kumar et al. ( , 2009) ) described that TCS decreased the production and/or secretion of P 4 , T, and E 2 in Leydig cells from Wistar rats in vitro and the level of hormones in the serum of male Wistar rats in vivo [34,35].Our data show that TCS increased the mRNA expression of Cyp17a1 but caused a decrease in the 17β-Hsd and Cyp19a1 mRNA expression.The enzymes encoded by these genes are involved in converting P 4 into T (Cyp17a1 and 17β-Hsd) and T to E 2 (Cyp19a1), which could explain the changes in level of the hormones in the cells.Moreover, the other cause of the reduced level of T in the cells may be the increased secretion of this hormone to the culture medium.As mentioned, honokiol is used as a neuroprotective agent [21].Our data show that honokiol as a partial PPARγ agonist prevents changes in Cyp17a1 mRNA expression and regulates P 4 release, which were disrupted by TCS.Interestingly, honokiol alone increased the P 4 production as well as the P 4 and T secretion.These data are consistent with the mRNA expression, as the decrease in the Cyp17a1 mRNA expression after the honokiol treatment correlated with the increase in the level of P 4 in the astrocytes.In our experiments, the full PPARγ agonist rosiglitazone alone decreased the T production and secretion, whereas in the co-treatment with TCS, it prevented the TCS-induced changes in the P 4 and T secretion and the E 2 production.Interestingly, the PPARγ antagonist GW9662 affected only the T production but prevented all TCS-induced changes in the neurosteroid production or secretion.Therefore, on the basis of our results, we believe that TCS is a PPARγ agonist, at least partially, or interferes with the PPARγ molecular pathway, which was particularly confirmed in the experiment with GW9662 (PPARγ full antagonist).We believe that the changes caused by honokiol may have been a result of the pleiotropic nature of this compound.Recent studies have described that honokiol interferes with the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, has neuroprotective potential in neuroinflammation [36], and modulates estrogen receptors [37], which may explain the differences between this compound and rosiglitazone.Moreover, in previous studies, resveratrol, which is a natural PPARγ agonist, decreased estradiol levels in bovine granulosa cells [38].Therefore, our data suggest that TCS interacts with the Pparγ molecular pathway.
Previous studies of TCS show that, despite the ROS-dependent mechanism of action, TCS mainly activates the AhR-dependent pathway [13].Moreover, some studies suggest that TCS can directly or indirectly affect the Nf-κb pathway [14], the mammalian target of rapamycin (mTOR) pathway [39], and the PPARγ pathway [40].Our present experiments show that, after the 24-h exposure, TCS increased the AhR mRNA expression but decreased the AhR protein level, which suggests the activation of this receptor and compensation effects at the mRNA level.Moreover, the partial Pparγ agonist (honokiol), full agonist (rosiglitazone), and antagonist (GW9662) alone did not affect the AhR protein level.The tool compounds did not affect the AhR decrease which was caused by TCS.Therefore, it can be concluded that the major TCS mechanism of action in astrocytes is AhR-dependent, and this is consistent with the current state of knowledge and previous models.
In our experiments, we did not observe a significant increase in the Pparγ protein expression in the mouse primary astrocytes.Interestingly, honokiol alone decreased the Pparγ mRNA expression, while an increase in the Pparγ mRNA expression was observed in the co-treatment with TCS.Moreover, in all the studied groups, a decrease in the Nf-κb mRNA expression was observed.To date, PPARγ agonists have been reported to inhibit Nf-κb expression [41].Crosstalk between PPARγ and NF-κB in human placental cells was described [42], during which NF-κB activation resulted in an increase in the PPARγ level.Moreover, similar to classic PPARγ agonists, honokiol can inhibit the NF-κB molecular pathway, which was demonstrated in the mouse MC3T3 cell line during osteoblastogenesis [43].Our previous studies showed that TCS increased Pparγ protein expression in mouse neocortical neurons but decreased Nf-κb expression [14].Therefore, given the multidirectional pleiotropic mechanism of honokiol action, it can be assumed that our data are consistent with the current state of knowledge, and TCS is a very weak Pparγ agonist or at least interferes with the Pparγ pathway in mouse astrocytes.
Sirtuins (Sirts) are nicotinamide adenine dinucleotide (NAD + )dependent deacetylases playing an important role in the control of metabolic processes [44].Sirt1 is localized in the nucleus and Sirt3 is found in mitochondria [45].Previous studies described that Sirt1 can interact with nuclear steroid hormone receptor co-activator proteins, i.e. p300, PPARγ, AhR, or PPARγ coactivator 1-alpha (PGC-1α) [46][47][48][49].Activation of these co-receptors results in the initiation of differentiation of muscle cells, adipogenesis, fat storage, and metabolism in the liver.Moreover, our previous studies showed that 10 µM TCS increased the Sirt1 and Sirt3 protein expression in mouse primary neurons after 24 and 48 h of exposure [18].In the present study, 1 µM TCS did not significantly change the Sirt1 protein expression but decreased the Sirt3 protein level in the primary mouse astrocytes.Honokiol increased the Sirt1 protein level, but the cell co-treatment with TCS and honokiol decreased the level of this protein, compared to the control.Interestingly, the astrocyte co-treatment with rosiglitazone and TCS as well as GW9662 and TCS increased the Sirt1 protein level.As reported by Han et al., PPARγ directly interacts with SIRT1 and inhibits SIRT1 activity in human lung fibroblasts, forming a negative feedback and self-regulation loop [50].Therefore, our data are consistent and confirmed that TCS at least partially acts through Pparγ in mouse primary astrocytes, which may be disrupted by honokiol.On the other hand, in our experiments the Sirt1 expression profile and the E 2 production were similar in all the experimental groups.To date, it has been described that E 2 production is affected by Sirt1 [51].Therefore, it cannot be excluded that this course of action and changes in the Sirt1 level may be a result of autocrine regulation.
In our experiments, TCS decreased the Sirt3 protein expression.A similar decrease in the Sirt3 protein expression was observed in the group treated with honokiol, rosiglitazone, and GW9662.Interestingly, in the group co-treated with honokiol and TCS, a slight increase in the Sirt3 level, compared to the TCS alone treatment, was observed.Moreover, in the group co-treated with GW9662 and TCS, no significant changes in the Sirt3 level was observed, compared to the control.To date, honokiol has been described to activate Sirt3 in primary cultures of cardiac myocytes prepared from neonatal rat hearts [52].Moreover, it has been reported that Sirt3 is a target of PGC-1α [53].Previous studies conducted by He et al. [54] demonstrated that AhR affects the Sirt3 level in the liver of C57BL/6 J mice [54].Therefore, in the case of the group co-treated with GW9662 and TCS, the absence of changes in the Sirt3 level is a result of inhibition of Pparγ by GW9662 and activation of AhR by TCS.Recent studies conducted by Matoba et al. (2024) showed that Sirt3 regulates proliferation and P 4 production in mouse Leydig cells [55].Matoba et al. also reported that Sirt3 knockdown resulted in 17β-Hsd mRNA expression and a decrease in progesterone production in Leydig cells [55].On the other hand, SIRT3 positively regulates the expression of folliculogenesis and luteinization-related genes as well as P 4 secretion in human luteinized granulosa cells [56].Similar to our data, Fu et al. showed that the decrease in the SIRT3 level results in 17β-HSD and CYP19A1 mRNA expression.It can be assumed that TCS and honokiol affect Sirt3 protein expression through different molecular mechanisms.The overlap of the two molecular pathways may explain the slight increase in Sirt3 in the group co-treated with honokiol and TCS.Therefore, it cannot be excluded that, in our model of endocrine-active cells i.e. astrocytes, the decrease in the Sirt3 protein level caused a decrease in the P 4 release.

Conclusions
Our data show that TCS in mouse primary astrocyte cultures can act as an EDC by destabilizing the production or secretion of P 4 , T, and E 2 .TCS affects the mRNA expression of enzymes involved in neurosteroidogenesis, i.e.Cyp17a1, 17β-Hsd, and Cyp19a1 (Fig. 5).Our data indicate that the partial PPARγ agonist honokiol prevents changes in Cyp17a1 mRNA expression caused by TCS.Similarly, honokiol inhibits TCS-stimulated P 4 release.However, rosiglitazone (classic PPARγ agonist) or GW9662 (PPARγ antagonist) had a much stronger effect.Therefore, we believe that the changes observed in the P 4 , T, and E 2 levels are a result of dysregulation of the activity of the abovementioned enzymes, whose expression can be affected by TCS through a Pparγ pathway.TCS causes a decrease in AhR and Sirt3 protein, which may be the result of the activation of these proteins.Due to the dysregulation of the production or secretion of neurosteroids in astrocytes, TCS reaching the brain may contribute to the development of neurodegenerative diseases in which an abnormal amount of neurosteroids is observed.

Fig. 1 .
Fig. 1.TCS toxicity in the primary mouse astrocyte culture.(A) LDH release level at increasing concentrations of TCS (1 nM-100 µM) in the primary mouse astrocyte culture after 24 h.(B) Effect of 1 and 10 µM TCS on Ki67 and p53 mRNA expression in the primary mouse astrocyte culture after 24 h.The statistical significance of each data point was analyzed by Tukey's test using oneway ANOVA for each study group; * p < 0.05, ** p < 0.01, and *** p < 0.001, compared to the control cells.

Fig. 2 .
Fig. 2. Impact of TCS on gene expression.The primary mouse astrocyte culture was exposed to 1 µM TCS, 10 µM Honokiol (HON), or HON with TCS for 24 h.After the exposure to the studied compound, the mRNA expression of the Nf-κb (A), Pparγ (B), AhR (C), Cyp17a1 (D), 17β-Hsd (E), Cyp19a1 (F) genes was determined.The statistical significance of each data point was analyzed by Tukey's test using one-way ANOVA for each study group; *** p < 0.001, compared with the control cells.## p < 0.01, and ### p < 0.001, comparison of the TCS-treated group with the group co-treated with TCS and HON.

Fig. 5 .
Fig. 5.The proposed mechanism of action of TCS.The scheme, summarizing the main findings in the presented study in the TCS-treated mouse astrocyte cells.Ttestosterone; P 4 -progesterone; E 2 -estradiol; The downwards, upwards, and left-right arrows were used to mark the decreasing, increasing, or no changes in the specific values, respectively.