Trichomonas vaginalis is a widespread and important sexually transmitted pathogen. Adherence to the surface of the host cell is the precondition for the parasitism and pathogenicity of this parasite. Trichomonas vaginalis adhesion protein 33 (TvAP33) plays a key role in the process of adhesion, but how this protein mediates the adhesion and pathogenicity of T. vaginalis to host cells is unclear.T. vaginalis were used to evaluate the pathogenicity of T. vaginalis. We constructed a complementary DNA library of VK2/E6E7 cells and screened the protein molecules interacting with TvAP33 by the yeast two-hybrid system. The interaction between TvAP33 and BNIP3 (Bcl-2 interacting protein 3) was analyzed by co-immunoprecipitation and colocalization.The expression of TvAP33 in trophozoites was knocked down by small interfering RNA. VK2/E6E7 cells and mice infected with T. vaginalis trophozoites adhering to VK2/E6E7 cells decreased significantly, and the inhibition of VK2/E6E7 cell proliferation and VK2/E6E7 cell apoptosis and death induced by T. vaginalis were reduced. Animal challenge experiments showed that the pathogenicity of trophozoites decreased following passive immunization with TvAP33 antiserum or blocking of the TvAP33 protein. Immunofluorescence analysis revealed that TvAP33 could bind to VK2/E6E7 cells. Eighteen protein molecules interacting with TvAP33 were identified by the yeast two-hybrid system. The interaction between TvAP33 and BNIP3 was further confirmed by co-immunoprecipitation and colocalization. When the expression of both TvAP33 and BNIP3 in trophozoites was knocked down by small RNA interference, the number of T. vaginalis adhering to VK2/E6E7 cells and the inhibition of VK2/E6E7 cell proliferation were significantly lower compared to trophozoites with only knockdown of TvAP33 or only BNIP3. Therefore, the interaction of TvAP33 and BNIP3 in the pathogenesis of T. vaginalis infecting host cells is not unique and involves other molecules.Following knockdown of TvAP33 expression, the number of T. vaginalis to host cells, providing a basis for searching for drug targets for T. vaginalis as well as new ideas for the prevention and treatment of trichomoniasis.Our study showed that the interaction between TvAP33 and BNIP3 mediated the adhesion and pathogenicity of The online version contains supplementary material available at 10.1186/s13071-023-05798-x. Trichomonas vaginalis is a widespread and important sexually transmitted pathogen in humans that causes a common infection of the urogenital system [T. vaginalis infection, the WHO estimated that this infection occurred in 6.3%  of women and 0.6% (95% UI 0.4–0.9) of men, with regional values ranging from 1.6% to 11.7% in women and from 0.2% to 1.3% in men. Clinical manifestations of T. vaginalis infection in women may include vulvovaginal irritation, lower abdominal pain, dyspareunia, dysuria and malodorous vaginal discharge [T. vaginalis infection [T. vaginalis increases the risk of human infection with human immunodeficiency virus (HIV) and mycoplasma [T. vaginalis infection support the need for more dynamic approaches to find new drug and vaccine targets.l system . In its Trichomonas vaginalis is an extracellular parasitic protozoan, adhering to the epithelial lining of the host's urogenital tract to survive. Following the adherence of T. vaginalis trophozoites to host cells, the ovoid free-swimming parasite transitions into a more elongated shape with accompanying pseudopodia, which results in the microenvironment of the colonization site of T. vaginalis being established. The niches continuously change as a result of different host biological processes and outside forces. Such changes include natural hormonal shifts that could lead to epithelial shedding in the female urogenital tract, excretion that could alter salinity, pH and toxicity of the environment and flux in communities of commensal and infectious microorganisms [T. vaginalis was observed not to change when T. vaginalis infected HeLa cells [T. vaginalis was able to recognize and specifically bind to host tissues. The adhesion of T. vaginalis to different cell types  has been previously reported [T. vaginalis infections [T. vaginalis cells (TvLPG) and that galectin-1 bound to T. vaginalis in a carbohydrate-dependent manner that was inhibited in the presence of TvLPG [rganisms , 9. HoweLa cells , indicatreported –15. Therfections , 17. Okuof TvLPG .T. vaginalis to host cells was reported to be mainly mediated by five T. vaginalis adhesion proteins (TvAPs) on the surface of trophozoites: TvAP23, TvAP33, TvAP51, TvAP65 and TvAP120. Southern blot analysis confirmed the existence of three single-copy TvAP33 genes and suggested a semiconservative genomic arrangement between T. vaginalis isolates [Trichomonas foetus, Mundodi et al. confirmed a role for TvAP33 as an adhesin in T. vaginalis [T. vaginalis cytoadherence by 40% [T. vaginalis cytoadherence but are also characterized as the α- and β-subunits of succinyl-CoA synthetase (SCS) that catalyze key steps of energy metabolism in T. vaginalis hydrogenosomes, are biotinylated in the cytosol and then imported exclusively into hydrogenosomes [Ureaplasma urealyticum infection [T. vaginalis infection [The adhesion of isolates . By usine by 40% . TvAP33 enosomes . van dernfection .T. vaginalis adherence to host cells, inhibiting the proliferation of host cells and inducing the apoptosis and death of host cells, which was achieved through interaction with receptor molecules on host cells.Some researchers believe that TvAPs are controversial adhesion molecules . In the 2 for 5 min; occasionally, cervical dislocation was used to confirm the effectiveness of euthanasia.All experiments performed in the present study were reviewed and approved by the Ethics Review Committee of Xinxiang Medical University (Reference No. XYLL-2018S006). We maximized our efforts to alleviate the distress and pain of the experimental animals as much as possible, and the infected mice were euthanized at humane endpoints when the mice appeared moribund. Euthanasia was predominantly performed by placing the experimental animal in a confined space and exposing it to 60–70% COT. vaginalis used in the present study was maintained in our laboratory and identified as actin genotype E by PCR restriction RFLP, which is the dominant genotype in the city of Xinxiang, Henan Province, China. As described in our previous studies [T. vaginalis was cultured in complete TYM medium in a humidified chamber maintained at 37 °C and 5% CO2. The parasites were collected by centrifugation and used for subsequent research.The strain of  studies , 26, T. 2. Cells of the 293T cell line were kept in our laboratory and cultured on Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% penicillin–streptomycin solution and 10% fetal calf serum in a humidified chamber at 37 °C and 5% CO2.Human vaginal epithelial cells (cell line VK2/E6E7) were purchased from American Type Culture Collection . The VK2/E6E7 cells were cultured on CnT-Prime Epithelial Cell Culture Medium  supplemented with 1% penicillin–streptomycin solution, in a humidified chamber at 37 °C and 5% COEight-week-old BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.  and kept under specific pathogen-free conditions.T. vaginalis trophozoites using the E.Z.N.A.™ Total RNA Kit I  according to the manufacturer’s instructions, and the genomic DNA was removed using DNase I. Complementary DNA (cDNA) was synthesized by reverse transcription PCR, and then the open reading frames (ORF) of TvAP33 (GenBank accession no. U87098.1) and glyceraldehyde 3-phosphate dehydrogenase  were amplified from the cDNA. The TvAP33 and GAPDH fragments were separately cloned into the pET-32a(+) vector to form recombinant plasmids pET-32a-TvAP33 and pET-32a-GAPDH, respectively, for subsequent research. The recombinant plasmids were then sequenced to confirm that the vectors were successfully constructed.Total RNA was extracted from Escherichia coli BL21(DE3) strain; when the optical density at 600 nm (OD600) of the culture reached 0.6 at 37 °C, isopropyl-b-D-thiogalactopyranoside  was added to the bacterial growth culture media to induce recombinant protein expression. The rTvAP33 and rGAPDH proteins were then purified by passage through a Ni2+-nitrilotriacetic acid (Ni–NTA) column , and the purified recombinant proteins were determined by 12% (w/v) sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). After the concentration was measured, the rTvAP33 and rGAPDH proteins were stored at − 20 °C for future study.The pET-32a-TvAP33 and pET-32a-GAPDH recombinant plasmids (rpET-32a-TvAP33 and rpET-32a-GAPDH) were introduced into competent For the generation of polyclonal antibodies (PcAbs), approximately 0.3 mg of the purified rTvAP33 and rGAPDH proteins were mixed with Freund’s complete adjuvant as a 1:1 mixture and subcutaneously injected into SD rats  in multiple places. Two weeks after the initial injections, a booster was given under the same conditions by using proteins that were mixed with Freund’s incomplete adjuvant as a 1:1 mixture. The rats were then reboosted three times at intervals of 1 week. Finally, the serum was collected, purified and stored until use. Sera collected before the injections of the proteins were used as negative sera.T. vaginalis trophozoites; the final concentration of transfected siRNA was set to 0, 50, 100, 150 and 200 nmol, and the transfection time was set to 0, 8, 12, 24 and 36 h.According to the gene sequence of TvAP33, three alternative small interfering RNAs (siRNAs) were designed to interfere with the expression of TvAP33 Table . The thr5 T. vaginalis trophozoites were inoculated into a 24-well plate, and 100 μl of transfection complex was added to each well. The cell culture plate was placed in a humidified chamber maintained at 37 °C and 5% CO2. After 4–6 h, the culture medium containing the transfection complex was replaced by fresh TYM medium, and T. vaginalis was cultured for a further 36 h.The 20 μmol of siRNA and 1.2 μl of Lipofectamine 2000 transfection medium  were diluted to working concentrations with 50 μl of OptiMEM medium (Thermo Fisher Scientific). The diluted siRNA and Lipofectamine 2000 were mixed evenly to form the transfection complex, which was allowed to stand at room temperature for 20 min. Approximately 5 × 10TM 5 system  with SYBR qPCR Master Mix . As described in the manufacturer’s instructions, the reaction mixture (20 μl) contained 2 × ChamQ Universal SYBR qPCR Master Mix (10 μl), forward primer , reverse primer , cDNA (2 μl) and ddH2O (7.2 μl). The amplification protocol consisted of a predenaturation step at 95 °C for 30 s; 40 cycles of a circular reaction at 95 °C 10 s and 60 °C for 30 s; and a melting curve at 95 °C for 15 s, 60 °C for 30 s and 95 °C for 15 s. The messenger RNA (mRNA) expression levels of the target genes, such as TvAP33, were normalized to those of the TvActin primers. Relative expression levels were calculated by the 2−△△Ct method.According to the gene sequences of TvAP33 and TvActin (GenBank accession no. KF747377.1), primers were designed according to the gene sequences of TvAP33 and TvActin (GenBank accession no. KF747377.1), respectively, and synthesized for detection by real-time PCR (qPCR) . The membrane was blocked with 5% (w/v) skim milk/phosphate buffered saline (PBS)-0.5% Tween 20 (PBST) and then incubated with specific anti-TvAP33 or anti-GAPDH rat antibody (1:100 dilution) for 1 h at 37 °C. The membrane was then washed 3 times with PBST for 5 min, and goat anti-rat immunoglobulin G (IgG)-horseradish peroxidase  was applied as secondary antibodies for 1 h at 37 °C. Finally, an enhanced chemiluminescence (ECL) kit  was used to visualize the bands on the basis of the operating manuals.Trichomonas vaginalis trophozoites were treated with TvAP33-siRNA and NC-siRNA and divided into the TvAP33 group and NC group, respectively. Trichomonas vaginalis without siRNA treatment was used as the T. vaginalis group. Three parallel controls were set in each group (n = 3). VK2/E6E7 cells were inoculated into a 24-well plate containing cell climbing slices. When the growth convergence of cells reached 80–90%, T. vaginalis in each group was labeled with CytoTrace™ Orange  and then used to infect VK2/E6E7 cells at a ratio of 1:1 (parasite:cell) for 15 and 30 min. The cell climbing slices were then fixed with 4% paraformaldehyde for 10 min at room temperature in the dark and subsequently examined by fluorescence microscopy . Five visual fields were randomly selected for each sample, and the number of trophozoites and cells was counted. In each field of vision, the adherence rate (in percentage) of T. vaginalis to host cells was calculated as follows:  × 100. The analyses were conducted based on data obtained from three individual experiments.5 cells/well) were inoculated into a 96-well plate. Trichomonas vaginalis trophozoites were treated with TvAP33-siRNA and NC-siRNA and divided into the TvAP33 group and NC group. Untreated T. vaginalis and uninfected VK2/E6E7 cells were used as the T. vaginalis group and blank group, respectively. The VK2/E6E7 cells were infected with T. vaginalis at a ratio of 1:1 (parasite:cell) for 12 h. Five parallel controls were used in each group (n = 5). After MTT solution (5 mg/ml) had been added to each well (20 μl/well), the VK2/E6E7 cells were cultured for a further 4 h at 37 °C and 5% CO2. The liquid and T. vaginalis were removed and the culture plates washed 3 times with PBS, following which 150 μl DMSO was added to each well, and the 96-well plate was shaken slightly for 10 min. Light absorption at 570 nm was measured on a microplate reader . The cell proliferation index was calculated as follows: /. The analyses were conducted based on data obtained from three individual experiments.VK2/E6E7 cells  for 12 h under conditions of 37 °C and 5% CO2, and then divided into the TvAP33 group, NC group and T. vaginalis group. Three parallel controls were set in each group (n = 3). The VK2/E6E7 cells were separated from the 24-well plate by trypsin and then resuspended in 500 μl of binding buffer after being washed twice with PBS by centrifugation at 500 g for 5 min. The supernatant was removed by centrifugation and resuspended in 100 μl of binding buffer. A 5-μl aliquot of Annexin V-FITC reagent (Sigma-Aldrich) and 5 μl of propidium iodide (PI) were added to the cell suspension, which was mixed and incubated at room temperature in the dark for 5 min. Samples of the cell suspension were measured by flow cytometry within 1 h. The independent experiment was repeated 3 times.As described above, the VK2/E6E7 cells were infected with T. vaginalis trophozoites at a ratio of 1:1 (parasite:cell) for 12 h under conditions of 37 °C and 5% CO2, and then divided into the TvAP33 group, NC group, T. vaginalis group and blank group. Five parallel controls were set in each group (n = 5). The cell culture medium and T. vaginalis were removed and the culture plates washed 2 times with PBS. PBS and 0.4% trypan blue were then added at a ratio of 9:1, and the cells were examined by optical microscopy (Nikon Corp.). Five visual fields were randomly selected for each sample, and the number of cells was counted. In each field of vision, the percentage cell viability was calculated as follows:  × 100. The analyses were conducted based on data obtained from three individual experiments.As described above, the VK2/E6E7 cells were infected with n = 15), and each mouse was intraperitoneally immunized with 200 μl of anti-rTvAP33 PcAb (200 μg/ml), rat normal IgG (200 μg/ml) or PBS. Mice in each group were immunized 4 times, with a 1-day interval between injections, and each mouse was intraperitoneally infected with 1 × 107T. vaginalis trophozoites after the first immunization. The survival of the mice was monitored throughout the month after the challenge, and animals showing symptoms euthanized by CO2. Thirty days later, the following formula was used to calculate the percentage survival rate of mice challenged with T. vaginalis: the number of surviving mice/the number of mice before immunization × 100.Forty-five BALB/c mice aged 6 weeks were randomly divided into three groups (n = 15). Trichomonas vaginalis trophozoites were blocked in anti-rTvAP33 PcAb diluted in PBS (anti-rTvAP65 PcAb:PBS = 1:5) at 37 °C for 15 min. Under the same conditions, the trophozoites of the other two groups were treated with normal rat IgG or PBS. The mice were intraperitoneally infected with blocked T. vaginalis trophozoites (1 × 107 trophozoites/mouse). The survival of mice was monitored throughout the month after the challenge, and animals showing symptoms were euthanized by CO2. Thirty days later, the following formula was used to calculate the percentage survival rate of mice challenged with T. vaginalis: the number of surviving mice/the number of mice before immunization × 100.Forty-five BALB/c mice aged 6 weeks were randomly divided into three groups  for 10 min at room temperature, permeabilized with 1% TritonX-100 in TBS for 10 min, washed 3 times in TBS containing 0.05% Tween-20 (TBST) and blocked with TBST containing 5% (w/v) bovine serum albumin (BSA) for 1 h at 37 °C. After three washes in TBST, TvAP65 antiserum (1:100 dilution) was added and to the well and incubated at 4 °C overnight. After a further three washes in TBST, the cells were maintained in darkness for 40 min in goat anti-rat IgG antibody  labeled with Cy3 diluted at 1:1000. After three washes in PBS, the fluorescent stain 4′,6-diamidino-2-phenylindole  was used to stain the nuclei for 15 min in darkness. After washing with TBST, fluorescent mounting medium (Beyotime) was added, and the cells were examined by fluorescence microscopy (Nikon Corp.).VK2/E6E7 cells were inoculated into a 24-well plate containing cell climbing slices. When the growth convergence of cells reached 90%, the cells were cocultured with the final concentration of 20 μg/ml of the TvAP33 recombinant proteins at 37 °C and 5% CO® II enzyme (Thermo Fisher Scientific). These recombinant plasmids were electroporated into E. coli DH10B, according to the following electrotransfer procedure: voltage 1500 V, resistance 200 Ω and capacity 25 μF. These bacterial solutions were diluted and coated onto solid medium, the number of bacterial clones was counted and the capacity of the library was calculated. The plasmid DNA was randomly extracted from 24 clones and restriction digested by SfiI to confirm the size of the inserts in the clones.The ORF of TvAP33 was cloned and inserted into the bait vector pDHB1 to form pDHB1-TvAP33. The cDNA library of VK2/E6E7 cells was constructed according to the instructions of the SMART cDNA Library Construction Kit . In brief, RNase-free DNase I  was used to remove the genomic DNA contamination from the prepared RNA samples. The mRNA was then isolated using the Oligotex mRNA Midi Kit . The purified mRNA was used to synthesize double-stranded cDNA, and both ends of the cDNAs were equipped with connectors for recombination into the prey vector. The cDNAs were purified and cloned into the pray plasmid pPR3-N with the BP ClonaseE. coli DH5α for amplification. The primers pPR3N-F and pPR3N-R were used to detect the gene fragments inserted in the selected prey plasmids by PCR. The isolated positive prey plasmids were retransformed into yeast NMY51 which contained the bait plasmid pDHB1-TvAP33 to verify the interaction between the two molecules. pTSU2-APP was used as a bait control, and pPR3-N or pNubG-Fe65 was used as the negative or positive prey control, respectively. The fragments inserted in these prey plasmids were sequenced, and the DNA sequences were matched in GenBank to obtain genetic information.The DUALhunter starter kit  was used to identify the binding molecule of TvAP33 from VK2/E6E7 cells. The bait plasmid pDHB1-TvAP33 was transformed into yeast NMY51. After confirmation of the expression of the bait and functional assay and optimization of the screening stringency, the plasmid pDHB1-TvAP33 was used to screen the proteins interacting with TvAP33 from the VK2/E6E7 cell cDNA library. Positive colonies were picked, and the plasmids were extracted by using the Yeast Plasmid Extraction Kit (Omega Bio-Tek). The selected prey plasmids were transformed into g for 2 min, following which the protein samples were analyzed by western blot.The eukaryotic expression vectors pDsRed-N1-TvAP33 and pCMV-3HA-BNIP3 (Bcl-2 interacting protein 3) were successfully constructed and cotransferred into 293 T cells . After 48 h, the cells were washed with PBS and harvested. Co-immunoprecipitation was performed by using the Pierce™ Co-Immunoprecipitation Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. In brief, the AminoLink Plus Coupling Resin was immobilized by 3.5 μg of affinity-purified anti-Flag antibody or mouse IgG as control (Thermo Fisher Scientific) at 37 °C for 2 h. Then, 350 μl of ice-cold IP Lysis Buffer and protein mixture (FLAG-TvAP33 and HA-BNIP3) from one 60-mm plate of transfected 293 T cells was added to immobilization resin and gently mixed for 2 h at room temperature. After washing, the bound samples were eluted in 60 μl of elution buffer via centrifugation at 2000 As described above, the colocalization of TvAP33 and BNIP3 was analyzed by immunofluorescence. The primary antibodies anti-TvAP33 and anti-BNIP3 were used to recognize TvAP33 and BNIP3, respectively.T. vaginalis trophozoites with knocked down TvAP33 expression, and then the adhesion of T. vaginalis to VK2/E6E7 cells, cell proliferation and cell activity were detected as mentioned above.The expression of BNIP3 was knocked down in VK2/E6E7 cells as described above. VK2/E6E7 cells with low expression of BNIP3 were infected with P < 0.05 indicated statistical significance.One-way analysis of variance (ANOVA) followed by Duncan’s multiple range test was performed to analyze the differences among different experimental groups. The survival-related data were analyzed by the Kaplan–Meier method. SPSS for Windows version 16 software  was used for the statistical analyses, and T. vaginalis trophozoites. The primers detecting the mRNA level of TvAP33 and the siRNA interfering with TvAP33 expression were screened by qPCR  was significantly lower than that on cells the T. vaginalis group (71.00 ± 3.81%) and the NC group (67.00 ± 3.54%). Similarly, after T. vaginalis infection for 30 min, compared with the adhesion rate of the T. vaginalis group (73.20 ± 4.32%) and the NC group (68.80 ± 2.55%), the adhesion rate in the TvAP33 group (27.40 ± 2.07%) was significantly reduced. These results indicate that TvAP33 played an important role in the adhesion of T. vaginalis to host cells.As shown in Fig. T. vaginalis, the proliferation index of VK2/E6E7 cells (0.65 ± 0.07) in the TvAP33 group was significantly higher than that of cells in the T. vaginalis group (0.51 ± 0.01) and NC group (0.51 ± 0.01). Flow cytometry detection of apoptosis  and the NC group . Under the microscope  and the NC group (74.08 ± 2.60%). These results showed that TvAP33 could inhibit cell proliferation and promote cell apoptosis and death during the process of T. vaginalis infecting host cells.We detected the proliferation of VK2/E6E7 cells by MTT assays  and PBS (10.60 ± 1.64 days), the survival rate of the mice infected with T. vaginalis blocked by anti-rTvAP33 PcAb (53.33%) was significantly increased, and the survival time (23.53 ± 7.24 days) was significantly prolonged.Similarly, as shown in Fig. T. vaginalis was weakened after TvAP33 was blocked.These results suggested that the pathogenicity of After VK2/E6E7 cells were cocultured with rTvAP33, we found that rTvAP33 (red fluorescence) could bind to VK2/E6E7 cells Fig. a, which 7 primary recombinants. The secondary library was obtained by recombining the plasmid of the primary library, and the recombination efficiency was 100%. In the secondary library, the average insert size was also > 1.0 kb, and the cDNA library contained 1.36 × 107 secondary recombinants. All metrics of this cDNA library conformed to the requirements, and this cDNA library could be used for subsequent screening.We successfully constructed the cDNA library of VK2/E6E7 cells by the gateway method . The identification results of the primary library indicated that the recombination efficiency was 96%, the average insert size was > 1.0 kb and the cDNA library contained at least 1.04 × 10The functionality and self-activation of the yeast two-hybrid system were tested, and the results showed that the bait pDHB1-TvAP33 and the screening system could be used to capture the molecules interacting with TvAP33 from the cDNA library of VK2/E6E7 cells  TvAP33 interacted with other molecules in VK2/E6E7 cells during the adhesion process.VK2/E6E7 cells with BNIP3 knockdown were infected by T. vaginalis with normal expression of TvAP33 was not significantly changed, compared with the group of VK2/E6E7 cells infected with T. vaginalis, while the proliferation in the group of VK2/E6E7 cells with BNIP3 knockdown infected by T. vaginalis with TvAP33 knockdown was significantly higher than that in the groups of VK2/E6E7 cells with normal expression of BNIP3 infected by T. vaginalis with TvAP33 knockdown. These results indicate that the interaction between TvAP33 and BNIP3 could inhibit cell proliferation after host cells were infected with T. vaginalis, the interaction between these two molecules was not irreplaceable and other molecules in VK2/E6E7 cells were involved.Similarly, as shown in Fig. T. vaginalis infection , iron and biliverdin . The actoperties . The int actions . HoweverT. vaginalis released microvesicle-like structures (MVs) [BNIP3 is a mitochondrial BH3-only member of the Bcl-2 protein family, and the protein is activated by hypoxia; it mainly functions as a cell death regulator under hypoxic conditions . BNIP3 aes (MVs) . These aes (MVs) . TherefoT. vaginalis to host cells and influencing the activity of host cells through an interaction with TvAP33. To address this issue, we knocked down the expression of BNIP3 in VK2/E6E7 cells and then analyzed the adhesion rate of trophozoites and the proliferation and viability of VK2/E6E7 cells after T. vaginalis infection of VK2/E6E7 cells with low BNIP3 expression. We found that the adhesion rates of T. vaginalis were not significantly changed after only knocking down BNIP3 in VK2/E6E7 cells, but when the expression of TvAP33 in T. vaginalis was knocked down, the adhesion rates of trophozoites on VK2/E6E7 cells with BNIP3 knockdown were significantly lower than those of trophozoites on VK2/E6E7 cells with normal BNIP3 expression; these results indicate that the interaction between TvAP33 and BNIP3 could positively affect the adhesion of T. vaginalis to host cells, but that the interaction was not irreplaceable and TvAP33 interacted with other molecules in VK2/E6E7 cells during the adhesion process. Recent studies have shown that BNIP3 can regulate many fundamental cellular functions, such as mitochondrial quality control, proliferation, differentiation and maturation [T. vaginalis to host cells by interacting with BNIP3 to reduce cell activity. In the present study, we found that compared with that of the group of VK2/E6E7 cells infected with T. vaginalis, the proliferation of VK2/E6E7 cells with BNIP3 knockdown infected by T. vaginalis with normal expression of TvAP33 was not significantly changed, while the proliferation in the group of VK2/E6E7 cells with BNIP3 knockdown infected by T. vaginalis with TvAP33 knockdown was significantly higher than that in the groups of VK2/E6E7 cells with normal expression of BNIP3 infected by T. vaginalis with TvAP33 knockdown. Similar to the role of BNIP3 in the adhesion of T. vaginalis to host cells, the interaction between TvAP33 and BNIP3 could inhibit host cell proliferation after host cells were infected with T. vaginalis, but the interaction between these two molecules was not irreplaceable, and other molecules in VK2/E6E7 cells were involved. BNIP3 could induce classical apoptosis through cytochrome c and caspase activation. However, the expression level of BNIP3 in VK2/E6E7 cells did not significantly change cell viability during T. vaginalis infection, and the results implied that TvAP33 did not induce host cell apoptosis and death through the BNIP3 pathway in T. vaginalis infection. Therefore, the molecular mechanism of TvAP33 reducing host cell activity in T. vaginalis infection needs further study.Since BNIP3, as a multifunctional protein, can interact with TvAP33, we investigated whether BNIP3 played a role in mediating the adhesion of turation ; thus, Bturation . On possT. vaginalis adheres to the epithelial lining of the host's urogenital tract to survive. The adherence to the surface of the host cell via TvAP33 is the precondition for parasitism and pathogenicity of this parasite. However, the molecular mechanism of adhesion and pathogenicity of T. vaginalis to host cells mediated by TvAP33 is unclear. In the present study, we knocked down the expression of TvAP33 in trophozoites by siRNA and found that TvAP33 played an important role in adhesion to host cells, inhibition of host cell proliferation and induction of host cell apoptosis and death during the process of T. vaginalis infection. The results of animal challenge experiments showed that the pathogenicity of trophozoites was decreased by passive immunization with TvAP33 antiserum or blocking of the TvAP33 protein. Immunofluorescence analysis showed that TvAP33 could bind to VK2/E6E7 cells, and 13 protein molecules interacting with TvAP33 were screened from the cDNA library of VK2/E6E7 cells by a yeast two-hybrid system. Moreover, the interaction between TvAP33 and BNIP3 was further confirmed by co-immunoprecipitation and immunofluorescence. Through T. vaginalis with low expression of TvAP33 infecting VK2/E6E7 cells with low expression of BNIP3, we revealed that the interaction between TvAP33 and BNIP3 could promote the adhesion of T. vaginalis to host cell and inhibit host cells proliferation, but the interaction was not irreplaceable, and other molecules in host cells were involved during the adhesion process. However, the interaction between these two molecules seemed to have no effect on host cell death induced by T. vaginalis.As an extracellular parasitic protozoan, T. vaginalis to host cells, although the role of the interaction of TvAP33 and BNIP3 in the pathogenesis of T. vaginalis infecting host cells was not unique. These results provide a basis for searching the drug targets of T. vaginalis and put forward new ideas for the prevention and treatment of trichomoniasis. In conclusion, our study demonstrated that the interaction between TvAP33 and BNIP3 mediated the adhesion and pathogenicity of Additional file 1: Figure S1. Amplification efficiency and specificity of TvAP33 and TvActin primers used for qPCR in this research. TvActin was used as internal reference gene in detecting the mRNA level of TvAP33 by qPCR. A Standard curve of primer amplification efficiency for qPCR. A1, TvAP33; A2, TvActin. B Melt curve of primers used for qPCR. B1, TvAP33; B2, TvActin.Additional file 2: Figure S2. The 3 alternative siRNAs. The mRNA level of TvAP33 after 3 alternative siRNAs interfered with T. vaginalis trophozoites. Asterisks indicate statistically significant difference at *P < 0.05, **P < 0.01; ns indicates absence of significance (P ≥ 0.05). TvAP33-siRNA1 was used to subsequently knock down the expression of TvAP33.Additional file 3: Figure S3. Optimization of the transfection conditions. A The siRNA concentration was optimized by transfecting siRNA labeled with fluorescent dye to T. vaginalis trophozoites. B The mRNA level of TvAP33 was analyzed by qPCR, after siRNA transfection of T. vaginalis trophozoites for 24 h. C The mRNA level of TvAP33 were analyzed by qPCR, after siRNA transfection of T. vaginalis trophozoites for 36 h. Asterisks indicate statistically significant difference at *P < 0.05, **P < 0.01; ns indicates absence of significance (P ≥ 0.05).Additional file 4: Figure S4. Quality inspection of the cDNA Library of VK2/E6E7 cells. A Agarose gel electrophoresis of total RNA in VK2/E6E7 cells. Lanes: M DNA molecular weight marker DL 5000 , 1 total RNA of VK2/E6E7 cells. B Agarose gel electrophoresis of mRNA in VK2/E6E7 cells. Lanes: M DNA molecular weight marker DL 2000 , 1 mRNA of VK2/E6E7 cells. C Agarose gel electrophoresis of the double-stranded cDNA. Lanes: M DNA molecular weight marker DL 2000 , 1 double-stranded cDNA. D Analysis of the recombination efficiency and the inserted fragment in primary library. Lanes: M DNA molecular weight marker DL 2000 , 1-24 PCR analysis of 24 bacterial colonies. E Analysis of the recombination efficiency and the inserted fragment in secondary library. Lanes: M DNA molecular weight marker DL 2000 , 1-24 PCR analysis of 24 bacterial colonies. F Identification of primary recombinants. G Identification of secondary recombinants.Additional file 5: Figure S5. Examination of the functionality and self-activation of yeast two hybrid system. A Verification of the functionality of yeast two hybrid system. The plasmids of PTSU2-APP and pNubG-Fe65 were used as positive bait vector and positive capture vector, respectively. The pPR3N plasmid was used to construct the cDNA library of VK2/E6E7 cells. B Examination of the self-activation of yeast two hybrid system. The plasmid of pOst1-NubI was used as positive bait control. The vector of pDHB1-TvAP33 was used to fish the protein molecules interacting with TvAP33 from cDNA library of VK2/E6E7 cells.Additional file 6: Figure S6. The screened molecules were co-transformed into yeast cells with bait TvAP33 to verify the interaction, respectively.Additional file 7: Figure S7. The 3 alternative siRNAs.Additional file 8: Figure S8. Amplification efficiency and specificity of BNIP3 and actin primers used for qPCR in this research. Actin was used as internal reference gene in detecting the mRNA level of BNIP3 by qPCR. A Standard curve of primer amplification efficiency for qPCR. A1, BNIP3; A2, actin. B Melt curve of primers used for qPCR. B1, BNIP3; B2, actin.Additional file 9: Figure S9. Optimization of the transfection conditions by transfecting siRNA labeled with fluorescent dye to VK2/E6E7. A Screening the optimal duration for transfecting siRNA into VK2/E6E7. A1, 6 h; A2, 12 h; A3, 24 h. B Screening the optimal siRNA concentration for transfecting siRNA into VK2/E6E7. B1, 50 nM; B2, 100 nM; B2, 150 nM.Additional file 10: Figure S10. Observation of T. vaginalis adherence to VK2/E6E7 cells under the fluorescence microscope (40×). The trophozoites were stained with red fluorescence by CytoTraceTM Orange.Additional file 11: Figure S11. Observation of VK2/E6E7 cells viability under the microscope (40×). These dead cells were stained with blue by trypan blue. A VK2/E6E7 cells, BT. vaginalis + VK2/E6E7 cells, CT. vaginalis + VK2/E6E7-NC-siRNA cells, DT. vaginalis + VK2/E6E7-BNIP3-siRNA cells, ET. vaginalis-NC-siRNA + VK2/E6E7 cells, FT. vaginalis-NC-siRNA + VK2/E6E7-NC-siRNA cells, GT. vaginalis-NC-siRNA + VK2/E6E7-BNIP3-siRNA cells, HT. vaginalis-TvAP33-siRNA + VK2/E6E7 cells, IT. vaginalis-TvAP33-siRNA + VK2/E6E7-NC-siRNA cells, JT. vaginalis-TvAP33-siRNA + VK2/E6E7-BNIP3-siRNA cells.


A ZnO@RuO2 composite in a 10:1 molar ratio was synthesized by microwave processing of a precipitate as a green, low-cost, and fast method, and then annealed at 300°C and 600°C to improve the catalytic properties. The physicochemical properties of the ZnO@RuO2 composites were investigated by X-ray powder diffraction (XRD), Raman and Fourier transform infrared (FTIR) spectroscopy, field emission scanning electron microscopy (FESEM), UV-Vis diffuse reflectance spectroscopy (DRS), and photoluminescence (PL) spectroscopy. The electrochemical activity of the samples was investigated by linear sweep voltammetry in acidic and alkaline electrolytes. We observed good bifunctional catalytic activity of the ZnO@RuO2 composites toward HER and OER in both electrolytes. The improved bifunctional catalytic activity of the ZnO@RuO2 composite by annealing was discussed and attributed to the reduced number of bulk oxygen vacancies and the increased number of established heterojunctions.Catalytic materials are the greatest challenge for the commercial application of water electrolysis (WEs) and fuel cells (FCs) as clean energy technologies. There is a need to find an alternative to expensive and unavailable platinum group metal (PGM) catalysts. This study aimed to reduce the cost of PGM materials by replacing Ru with RuO ZnO is an attractive photo-electro catalyst due to its wide-band gap energy at room temperature (3.37 eV), which can be tuned by metal and non-metal ion doping, incorporation of crystalline defects in the form of vacancies and interstitials, modification of particle morphology and surface chemistry, etc. 2 onto RuO2 and microwave processing of the precipitate were employed to reduce the number of oxygen vacancies and improve local crystal symmetry. To our knowledge, no other published data have described the same processing of ZnO@RuO2 composite particles. In this study, we used ZnO:RuO2 in a 10:1 molar ratio. The as-prepared 10ZnO@RuO2 composite was additionally annealed at 300°C and 600°C to further reduce the number of bulk oxygen vacancies. We investigated the crystal structure, morphology, and optical and photo-electro catalytic properties of the processed 10ZnO@RuO2 catalyst particles in detail. We discussed the possibilities of using ZnO@RuO2 composites as photo-electro catalysts for HER, OER, and ORR in both alkaline and acidic solutions.We previously demonstrated the promise of ZnO-based nanostructured materials as photo-electro catalysts for OER and bifunctional catalysis for OER/ORR in moderately alkaline solutions . The pre2, purity >99.5, Lach-Ner, Neratovice, Czech Republic) and ruthenium oxide  were used as zinc and ruthenium sources, respectively. Sodium hydroxide  was used as a precipitating agent. All chemicals were used as supplied by the manufacturers, without additional purification. Distilled water was used as a solvent and dispersant and to rinse the powder, while absolute ethanol  was used for the final rinse.Zinc chloride (ZnCl2 in a 10:1 M ratio was prepared by microwave processing of an in situ precipitated Zn(OH)2 on RuO2. For this purpose, 0.044 g of RuO2 was dispersed in 100 mL of distilled water under constant stirring with a magnetic stirrer. After stirring for 5 min, 0.8976 g ZnCl2 was added to the RuO2-water dispersion. Next, 20 mL of 1.75 M NaOH was added dropwise to the mixture of dispersed RuO2 and zinc solution with constant stirring. After stirring at 50°C for 90 min in total, the as-prepared black precipitate was microwave processed in a domestic oven  for 5 min. After cooling to room temperature, the precipitate was centrifuged at 5,000 rpm for 10 min, rinsed five times with distilled water, and then with absolute ethanol to remove the surface residues of the starting chemical solutions. The synthesized powder was dried in an oven at 80°C for 24 h.A composite of ZnO and RuO2 composite. The analysis was determined by simultaneous TG-DTA  between 30°C and 800°C under an airflow of 20 mL/min in an Al2O3 pan. The heating profile was as follows: the material was stabilized at 30°C for 5 min and then heated to 800°C at 10°/min. From the TG-DTA, curves 300°C and 600°C were selected as further annealing temperatures. The composite was annealed in a tube furnace in an air atmosphere at a heating rate of 10°/min and a dwell time of 1 h.To vary the particle crystallinity and the number of oxygen vacancies, the composite sample was annealed at two different temperatures. The annealing temperatures were selected based on thermogravimetric and differential thermal analysis (TG-DTA) of the as-prepared ZnO@RuO2, 10ZnO@1RuO2-300, and 10ZnO@1RuO2-600, where the numbers 10 and 1 signify the molar ratio of ZnO and RuO2 and 300 and 600 signify the annealing temperatures.This study denotes the prepared photo-electro catalysts as 10ZnO@1RuO–1 with a resolution of 4 cm–1. XRD data were collected at room temperature using a Philips PW 1050 diffractometer with CuKα1,2 (λ = 1.54178 Å) Ni-filtrated radiation. The diffraction intensity was measured from 20° to 80° 2θ, with a step size of 0.05° and a counting time of 5 s per step. The working conditions were 40 kV and 20 mA. The crystal phases were identified using Mach!3 database software (D) were calculated from XRD line-broadening using the Scherrer equation  with a resolution of 4 cm−1. The 532 nm line of a diode-pumped solid-state high-brightness laser was used as the excitation source. Conventional and high-resolution transmission electron microscopy (TEM/HRTEM) analyses were performed on an FEI Talos F200X microscope  at 200 kV. Samples for TEM analysis were prepared by dispersing the powders in ethanol by ultrasound. After dispersion, a drop of the solution was placed on a carbon-coated copper grid and dried in air. The recorded TEM micrographs were used to estimate the mean particle size by measuring 100–200 particles using ImageJ software . The particle morphologies were characterized by field emission scanning electron microscopy . Samples for FE-SEM analysis were dispersed in water in an ultrasonic bath for 30 min; after dispersion, a few drops were filtered through a cellulose acetate membrane. The membrane was placed on carbon tape on the aluminum stub and coated with carbon for electron reflection. Before analysis, the samples were vacuumed for 15 min. Energy-dispersive X-ray fluorescence (EDX) spectroscopy with elemental maps was performed on an FEI SCIOS 2 Dual Beam electron microscope  operated at 10 kV. UV-Vis DRS were recorded using an Agilent Cary 5000 spectrophotometer equipped with a diffuse reflectance accessory. The measurements were performed in the range of 800–200 nm with a data interval of 1 nm and a scan rate of 600 nm/min, using a commercial PTFE standard for baseline correction. The PL spectra were recorded on a Horiba Jobin Yvon Fluorolog FL3–22 spectrofluorometer using Xe lamp excitation.The Fourier transform infrared (FTIR) spectrum was recorded using a Thermo Scientific™ Nicolet™ iS™10 Spectrometer equipped with an attenuated total reflectance (ATR) accessory. The measurements were performed in the wavenumber range of 400–4,000 cmsoftware , with resoftware . The unisoftware , while cequation . The μ-R2SO4 (p.a. Merck), pH ∼ 1, was used as the acidic electrolyte, while 0.1 M NaOH (p.a. Merck), pH ∼ 13, was used as the alkaline electrolyte. The working electrode with a surface area of about 0.3 cm2 was coated with catalyst ink prepared by mixing 5 mg of a catalyst powder with 10 μL of 5% Nafion solution  as a binder, 50 μL ethanol, and 50 μL water. This slurry was homogenized in an ultrasonic bath for 45 min. Subsequently, 5 μL of the prepared ink was coated as a thin film on the working electrode. To evaporate the solvent, the electrode was dried for 45 s with an infrared lamp (Tungsram INFRASEC 250 W). Measurements were performed in a potential range from 0.2 to 1.9 V vs. SCE for the OER reaction and from 0.2 to −1.9 V vs. SCE for the HER reaction with a scan rate of 20 mV⋅s−1. The measurements were performed in the dark and under illumination, after 60 min of exposure to an Osram Ultra-Vitalux 300 W lamp. The distance between the cell and the light source was set to 15 cm, which resulted in a light intensity of approximately 100 mW⋅cm−2.The electrocatalytic (EC) activities of the prepared samples were tested using linear sweep voltammetry (LSV). The EC measurements were performed on an Ivium VertexOne potentiostat/galvanostat in a conventional three-electrode quartz cell consisting of a glassy carbon as a working electrode, a platinum foil as a counter electrode, and a saturated calomel electrode (SCE) as a reference electrode. The tests were carried out in both acidic and alkaline electrolytes. An aqueous solution of 0.1 M H2 gas (99.998%) was bubbled into the electrolyte solution for 15 min before and during the electrochemical measurements. The LSV was measured in the potential range between 0.2 and 1.2 V vs. RHE with a scan rate of 5 mV⋅s–1. The overpotential (η) was defined as μ = E (vs. RHE)–1.23 V. The number of electrons transferred in the ORR process was calculated using the Koutecky–Levich equation . The LSV measurements were performed in an O2-saturated 0.1 M NaOH aqueous solution at different rotation speeds  at a scan rate of 10 mV s−1 from 0.2 to 1.2 V vs. RHE.All potentials were measured against the SCE and converted to a reversible hydrogen electrode (RHE) scale using Eq. 1 M NaOH . The samη is the overpotential, b is the Tafel slope (mV⋅dec−1), j is the current density, and jo is the exchange current density (mA⋅cm−2).The electrochemical reaction kinetics were analyzed using Tafel plots based on the Tafel equation Eq. :η=b⋅logCdl) using Eq. Cs is the specific capacitance of the catalyst; usually 0.035 mFcm–2 and 0.040 mFcm–2 in 1 M H2SO4 and 1 M NaOH, respectively  for all electrocatalysts in both electrolytes. CVs were performed in a non-Faradaic region at seven different scan rates . The average current densities (Δj = (ja−jc)/2) were plotted as a function of the scan rate, and Cdl was determined as the slope of the linear fit  was determined by the electrochemical double-layered capacitance electrocatalytic activity, in which the annealing temperature has a large impact . To dete2 composites, 2. The XRD pattern of the pristine ZnO indicates a pure, highly crystalline, wurtzite-type structure with a hexagonal phase , while the diffraction peaks in the XRD pattern of bare RuO2 are a rutile-type structure with a tetragonal phase . The ZnO@RuO2 composite powder consisted of hexagonal ZnO and tetragonal RuO2 without other crystal phases. According to the XRD patterns, the composites annealed at 300°C and 600°C had the same phase compositions as the as-prepared ZnO@RuO2 composite, but with slightly increased crystallinity from 92.15% for ZnO@RuO2-300% to 94.5% for ZnO@RuO2-600, which correlated with the weight loss of about 0.6% accompanying the large exothermic peak from 300°C to 600°C in the TG-DTA graphs . For comparison, besides the XRD patterns of the ZnO@RuOA graphs .2, and the prepared composites are listed in 2 powders agree well with the values reported in the corresponding COD cards . The calculated values showed that additional annealing caused a reduction of the ZnO and RuO2 crystal lattices in the ZnO@RuO2 composite. Moreover, the results indicated an increase in the average crystallite size of the zinc oxide after 1 h of annealing at 300°C and promoted growth at 600°C, while the average crystallite size of RuO2 was slightly reduced during annealing at 600°C. In addition, the crystallinity increased with increasing annealing temperature.The calculated unit cell parameters, average crystallite size, and degree of crystallinity of pristine ZnO, bare RuO2+ in the coordination IV equal to 0.74 Å  mode, indicated that the ZnO crystallites did not exhibit elongation in the c-axis direction typical of ZnO particles  mode consisting of A1 (at 574 cm–1) and E1 (at 587 cm–1) modes, while the other at 635 cm–1 occurred due to a combination of acoustic and optical modes (TA + LO). The A1 (LO) mode was accompanied by a broad band of low intensity near 480 cm–1, denoted as 2LA, which was ascribed to the interfacial surface phonon mode, characteristic of surface defects (1 (LO). The relatively high-intensity A1 (LO) + E1 (LO) modes in the Raman spectrum of pristine ZnO  planes of ZnO, while the d spacing of 2.55 Å corresponds to the (101) planes of RuO2. The TEM analysis showed that the ZnO particles . Furthermore, additional annealing of the composite had a negligible effect on the percentage of reflectance in the spectral range of 380–700 nm. The Kubelka–Munk function was applied to determine the direct and indirect band gap energies (Ebg) of the catalyst particles (F(R) × E]2, i.e., [F(R) × E]1/2 with respect to E (eV) to 0, 2, 10ZnO@1RuO2, 10ZnO@1RuO2-300, and 10ZnO@1RuO2-600 were 3.32, 1.57, 3.30, 3.26, and 3.24 eV, respectively. The estimated values of the indirect band gap energies of ZnO, RuO2, 10ZnO@1RuO2, 10ZnO@1RuO2-300, and 10ZnO@1RuO2-600 were 3.20, 0.60, 3.16, 3.15, and 3.11 eV, respectively.The optical properties were studied by UV-Vis diffuse reflectance (DRS) and photoluminescence (PL) spectroscopy . Figure articles . These e2 crystal lattice. The PL emission spectra of all the catalysts studied 2 precipitate via microwave irradiation  had a significantly lower number of bulk defects in the crystal structure. Moreover, additional annealing of the as-prepared 10ZnO@1RuO2 composite significantly affected the bulk defects, indicating that increased annealing temperature reduced the number of bulk oxygen vacancies to a greater extent.Room-temperature PL spectroscopy was used to study the effects of annealing temperature on point defects in the 10ZnO@1RuO studied  showed a studied . The low studied  indicate studied . The mosadiation . In contThe synthesized catalysts were used to prepare (photo)electrodes, which were analyzed as both (photo)anode and (photo)cathode depending on the applied potential interval. The overall water-splitting reaction can be expressed as two half-reactions, the mechanism of which depends on the reaction conditions, i.e., on the nature of the electrolyte  :In an acidic electrolyte:In an alkaline electrolyte:2. −1. The estimated onset potentials at 10 A g−1 for OER in alkaline solution were 2.08, 2.06, 2.25, 2.36, and 1.94, for ZnO, 10ZnO@1RuO2, 10ZnO@1RuO2-300, 10ZnO@1RuO2-600, and RuO2, respectively. The same activity was observed when the current density values were compared at 2.9 V vs. RHE: 36.1, 92, 55.6, 38.7, and 175.7 A g−1 for ZnO, 10ZnO@1RuO2, 10ZnO@1RuO2-300, 10ZnO@1RuO2-600, and RuO2, respectively. The same trend of catalytic activity was also observed for HER in the 0.1 M NaOH solution. ZnO showed low electrocatalytic activity  while the onset potentials at −10 A g−1 of 10ZnO@1RuO2, 10ZnO@1RuO2-300, 10ZnO@1RuO2-600, and RuO2 were −0.289, −0.671, −0.284, and −0.251 V vs. RHE, respectively. The current densities at 0.9 V vs. RHE were 5.9, 65.1, 18.9, 103.2, and 84.7 mA cm-2 for ZnO, 10ZnO@1RuO2, 10ZnO@1RuO2-300, 10ZnO@1RuO2-600, and RuO2, respectively.Two half-reactions, HER and OER, are commonly studied in alkaline and acidic media . Many pu2-600 catalysts, where the onset potential was reduced by 0.163 V. For OER, the onset potential shifted by a similar value in the 10ZnO@1RuO2-300 sample. The same trend was observed for the photocurrent. The 10ZnO@1RuO2-600 sample showed a current of 175.9 Ag−1 in the case of HER and 134.9 Ag−1 for OER at a potential of 2.9 vs. RHE.After 60 min of light irradiation, during the analysis of HER and OER in 0.1 M NaOH, the onset potentials for all samples shifted to lower values and the current densities increased. This indicated that photocarriers were formed in the catalysts after light exposure, which enhanced the reduction process of the adsorbed proton . The mos2SO4 solution were 2.15, 2.21, 2.14, and 1.97 V vs. RHE for 10ZnO@1RuO2, 10ZnO@1RuO2-300, 10ZnO@1RuO2-600, and RuO2, respectively. For ZnO, the onset potential was 2.196 V vs. RHE at 6.7 A g−1. Comparisons of current density values at 2.2 V vs. RHE showed similar activity in the dark for all composite samples (13 Ag−1). Upon light irradiation, the onset potential of the annealed composites, 10ZnO@1RuO2-300 and 10ZnO@1RuO2-600, shifted to lower values (0.180 V), while the onset potential of the as-prepared 10ZnO@1RuO2 composite decreased to approximately 0.140 V. The highest photocurrent of 41.1 Ag−1 was observed for 10ZnO@1RuO2-600. Regarding HER in the 0.1 M H2SO4 solution, the onset potential at −10 A g-1 were −0.714, −0.689, −0.537, and −0.279 V vs. RHE for ZnO, 10ZnO@1RuO2, 10ZnO@1RuO2-300, 10ZnO@1RuO2-600, and RuO2, respectively.Regarding the OER, in addition to the number of active sites on the electrocatalyst surface, the oxidation state of these active sites also plays an important role, with high oxidation states favoring OER . In the 2-600; the values obtained are −0.537 V vs. RHE and 51.2 Ag−1, respectively. All the samples show improved photocatalytic activity; in particular, both annealed composites had lower onset potentials of about 0.160 V, while the onset potential of the as-prepared composite shifted to a lower value of 0.188 V. The highest photocurrent of about 117.8 Ag−1 was observed for 10ZnO@1RuO2-600. The Tafel slope is an important parameter for evaluating the electrocatalytic kinetics of HER and OER reactions, in which a smaller Tafel slope indicates better electrocatalytic behavior for each of the reactions (η vs. logj)  and light (∼130 mV⋅dec−1) experimental conditions, while OER in the alkaline solution had the slowest reaction. The Tafel slopes also showed that the OER was much faster in the acidic electrolyte , while the HER was slightly slower in an acidic electrolyte compared to the HER in an alkaline medium, especially under light irradiation (∼250–380 mV⋅dec−1). The theoretical values of the Tafel slope for HER were 30, 40, and 120 mV dec−1 corresponding to Tafel, Heyrovsky, and Volmer reactions, respectively (2-600 was a good photo(electro) catalyst for HER in the alkaline and acidic mediums, as well as the OER in the acidic medium. The results of the comparisons of catalytic activities of different ZnO-based materials in terms of the onset potentials and Tafel slopes are listed in Regarding the catalytic activity toward HER, the lowest onset potential and the highest current are also registered for 10ZnO@1RuOeactions . Thus, ts. logj)  based ons. logj) . The Tafectively . In the ectively . The onsEOER/HER) between the onset potential for OER and HER, where the smallest ΔEOER/HER value indicates the better bifunctional electrode. Under light irradiation conditions, the smallest ΔEOER/HER was obtained for 10ZnO@1RuO2-600 catalysts; the values of 2.34 V and 2.11 V in H2SO4 and NaOH, respectively, suggest their potential for bifunctional photoelectrode materials. In the dark condition, the same catalysts showed the smallest potential variation in HER/OER in the acidic solution, compared to 10ZnO@1RuO2 in the alkaline solution. The best bifunctional catalytic activity of 10ZnO@1RuO2-600 among all the evaluated catalysts can be explained by the synergy of the reduced number of bulk oxygen vacancies . 2 at a rotation rate of 200–1,600 rpm and a scan rate of 5 mV⋅s−1. As shown in The ORR activity of the catalysts was tested in both alkaline and acidic electrolytes. None of the tested catalysts showed good catalytic activity for ORR in the acidic electrolyte, while 10ZnO@1RuOThe mechanism of ORR in alkaline electrolytes can follow different pathways depending on the number of electrons; it can be a direct four-electron pathway Eq. , 8 Mili:O2+2 H2j−1 vs. ω−1/2) at different electrode potentials were constructed to calculate the electron transfer number (n) based on the Koutecky–Levich (K–L) equation /2) were plotted as a function of the scan rate 2 precipitated on commercial RuO2 particles in a 10:1 molar ratio. To determine the influence of annealing temperature on the photo-electro catalytic activity of the 10ZnO@1RuO2 composite, influential properties such as phase composition, crystallinity, Zn, Ru, and O elemental distribution, morphology, optical properties, and band gap structure were studied in detail. XRD and Raman spectroscopy revealed that additional annealing of 10ZnO@1RuO2 composite led to increased average crystallite size, shrinkage of both unit ZnO and RuO2 cells in the composite, and increased crystallinity with enhanced local (short-range) ordering in ZnO. The EDS mapping showed a homogenous distribution of the Zn, Ru, and O elements in the composite particles. UV-Vis DRS and PL spectroscopy revealed that a small amount of RuO2 in the 10ZnO@1RuO2 composite significantly increased the absorption in the visible region and reduced the number of bulk oxygen vacancies. The LSV studies showed that, compared to pristine ZnO, all investigated 10ZnO@1RuO2 composites showed enhanced catalytic activity toward HER in both alkaline and acidic solutions. Moreover, in the alkaline solution, 10ZnO@1RuO2-600 exhibited better activity toward HER than bare RuO2. 10ZnO@1RuO2-600 also showed the best catalytic activity toward OER in the acidic solution. In the alkaline solution, the as-prepared composite showed better catalytic activity toward OER than both annealed composites. The improved HER and OER activity of the composites compared to pristine ZnO can be explained by the synergy of 1) improved electron transfer due to the metallic conductivity i.e., the low resistivity of RuO2, 2) the reduced number of bulk oxygen vacancies, which were further reduced by the composite annealing, and 3) the increased number of established heterojunctions, which modulated the electronic structures, increased the number of active sites, and accelerated electron transfer. The photo-electro catalytic activities of the ZnO-based composites were significantly improved by the addition of a small amount of RuO2 nanoparticles, which absorbed almost 100% of visible light. The low activities of the 10ZnO@1RuO2 composites toward ORR were additional confirmation that ORR preferred catalysts with sufficient surface oxygen vacancies.This study presented the results of the detailed physicochemical characterization of 10ZnO@1RuO


Ceratitis capitata, in citrus crops in Spain. However, the sustainable use of this insecticide is compromised by the detection of spinosad resistant alleles in field populations, which may lead to control failures in the future. Mutations, generating premature stop codons at the gene coding for the target alpha 6 subunit of the nicotinic acetylcholine receptor of C. capitata (Ccα6), have been associated with spinosad resistance in both laboratory strains and field populations. In this work, we showed that full-length transcripts from individuals carrying wild-type isoforms of the gene are translated into Ccα6 proteins that locate in the membrane of the brain cells, while truncated transcripts from spinosad resistant strains could be translated into truncated Ccα6 that, for the most part, are unable to reach their expected functional destination in the membrane. We proposed that the difference of location of Ccα6 observed in spinosad resistant strains is probably determining its resistant phenotype. In addition, we provide a tool for immunodetection of truncated forms of Ccα6, that can be useful for resistance management programs.The insecticide spinosad is widely used for the control of the Mediterranean fruit fly (medfly), α6 gene of Ceratitis capitata flies have been previously associated with spinosad resistance, but it is unknown if these transcripts are translated and if so, what is the location of the putative truncated proteins. In this work, we produced a specific antibody against C. capitata α6 (Ccα6) and validated it by ELISA, Western blotting and immunofluorescence assays in brain tissues. The antibody detects both wild-type and truncated forms of Ccα6 in vivo, and the protein is located in the cell membrane of the brain of wild-type spinosad sensitive flies. On the contrary, the shortened transcripts present in resistant flies generate putative truncated proteins that, for the most part, fail to reach their final destination in the membrane of the cells and remain in the cytoplasm. The differences observed in the locations of wild-type and truncated α6 proteins are proposed to determine the susceptibility or resistance to spinosad.The α6 subunit of the nicotinic acetylcholine receptor (nAChR) has been proposed as the target for spinosad in insects. Point mutations that result in premature stop codons in the  Ceratitis capitata, one of the main pests of citrus and other fruits worldwide, is not an exception, and resistance to some of the insecticides used for its control has already been detected in field populations, as to malathion /CyO, which expresses GAL4 under the control of nAChRalpha6 regulation; stock Dm 25835: y [1]v [1];P{y[+t7.7]v[+t1.8] = TriP.JF01853}attP2, which expresses dsRNA for RNAi of nAChRalpha6 (FBgn0032151) under UAS control in the VALIUM10 vector; stock Dm 57818: y [1]v [1];P{y[+t7.7]v[+t1.8] = TriP.HMJ21826}attP40/CyO, which expresses dsRNA for RNAi of nAChRalpha6 (FBgn0032151) under UAS control in the VALIUM20 vector; stock BL 52676: w [1118]; P{y[+t7.7] w[+mC] = GMR27E08-lexA}attP40, which expresses lexA under the control of DNA sequences in or near elav; stock BL 66545: w[*]; P{w[+mC] = LexAop-CD8-GFP-2A-CD8-GFP}2; TM2/TM6B, Tb [1], which expresses membrane-localized CD8-GFP under the control of LexAop. All of them were kept in the laboratory without exposure to insecticides. Flies were kept in standard fly food at 25 °C, unless otherwise indicated. To analyze the effectivity of the dsRNAs for RNAi, males from the stocks carrying the dsRNA of nAChRalpha6 (Dm 25835 and Dm 57818) were crossed with virgin females from the stock expressing GAL4 under the control of nAChRalpha6 promoter (Dm 84665). Pooled crosses were established to obtain the genotypes RNAi−25835nAChRα6-gal4 > UAS-nAChRα6 [α6-dsRNA (1)] and RNAi−57818nAChRα6-gal4 > UAS-nAChRα6 [α6-dsRNA (2)], both producing the nAChRalpha6 dsRNA, distinguishable by not showing the CyO phenotype but the wild-type. Likewise, BL 52676 and BL 66545 flies were crossed to generate w; elav-LexA>LexAop-CD8GFP F1 genotypes which express membrane-localized CD8-GFP and wild-type Dα6. The resulting F1 flies were collected and used for immunofluorescence assays . The isoform was amplified in a volume of 250 μL using 0.4 μM of forward alpha6-NtermF (5′-CGCATATGGACCCGTCGCTGTTTG) and reverse alpha6-NtermR (5′-GCGTCGACCTAGGGCACGTACAGAC) oligonucleotides, 2 U of AmpliTaq Gold DNA Polymerase , 10× PCR Buffer II, 1.5 mM MgCl2, 0.2 mM dNTPs , and a colony carrying the specific isoform. PCR conditions were as follows: an initial denaturation step at 95 °C for 10 min; 40 cycles of 95 °C for 30 s, 65 °C for 30 s, and 72 °C for 40 s, with a final step of 72 °C for 10 min for full extension. PCR products were analyzed by electrophoresis on 1.2% agarose gel . The bands of interest were purified from the gel with NZYGelpure  following the manufacturer’s instructions, and the Ccα63AQ68*region was then sequenced . PCR products were cloned into the pGEM-T-easy vector following the manufacturer’s instructions. Transformation of DH5α cells  was performed using a standard heat shock protocol and cultures were established. Plasmids were purified , eluted in 100 µL and sequenced . Clones were digested with NdeI and SalI  and fragment-size was checked by electrophoresis on 1.2% agarose gel. Digested fragments were cloned into the pET12a following the manufacturer’s instructions and DH5α were transformed by heat shock, cultures established, and fragment-size checked by NdeI and SalI digestion and electrophoresis. Clones for Ccα63AQ68* were transformed into BL21(DE3) cells  by heat shock, cultures established, and plasmids purified, while the presence of the fragment was checked by digestion and electrophoresis.The truncated isoform Ccα6 Q68 see , was gen® TGXTM Precast Protein Gels, Bio-Rad, Spain) was performed to analyze the protein extracts. Once the induction of the expression was checked, preparative gels of electrophoresis were performed to separate the specific band, which was then homogenized  and centrifuged at 4000 rpm for 10 min twice, discarding the pellet. Supernatant was lyophilized over-night. It was then resuspended in distilled water and dialyzed for 90 min. Purified Ccα63aQ68* was sequenced  to obtain the N-terminal sequence.Transformed BL21(DE3) cells were grown in LB medium with ampicillin (150 mg/mL) at 37 °C and 200 rpm for 6 h, IPTG was then added to a final concentration of 2 mM and cultures were cultivated over-night. Cultures without IPTG were used as non-induction controls. Cultures were centrifuged at 4000 rpm for 10 min at 4 °C and pellets were resuspended and sonicated for 2 min. Tubes were centrifuged at 10,000 rpm for 10 min at 4 °C. Soluble and non-soluble fractions were separated. SDS-PAGE in a 4–20% polyacrylamide gel  at the Proteomics’ department of the National Center for Biotechnology .The peptide Ccα6 (N-term KESCQGPHEKRLLNHLLSTYNTLER C-term) was designed from the sequence of Immunization was performed on female New Zealand rabbits at the Animal Facility of the CIB Margarita Salas. The purified synthetic peptide Ccα6 was used as antigen at a concentration of 1 mg/mL. It was emulsified with complete Freund’s adjuvant (1:1) for the first injection, and with incomplete Freund’s adjuvant (1:1) for the second and third injections. Injections were spaced three weeks apart. A pre-immune blood sample was extracted before the first immunization as a control. Blood samples were centrifuged at 4 °C and 4000 rpm for 10 min, and supernatants were aliquoted and stored at −20 °C. The anti-Ccα6 antibody was purified from the serum sample using HiTrap NHS-activated HP columns  coupled with the purified synthetic peptide Ccα6, following purification and coupling procedures using a syringe described in the manufacturer’s Antibody Purification Handbook. Elution was achieved by lowering the pH to 2.5.TM MicroWellTM 96 well solid plates, Sigma-Aldrich, St. Loius, MO, USA) and wells were washed with distilled water between steps. First, wells were coated with 5 µg/mL of Ccα6 antigen in Na2CO3  and incubated over-night at 37 °C. Residual action spots were blocked with BSA 1% in PBS for 1 h at 37 °C. The antibody was diluted in BSA 0.5%, added to the corresponding wells, and incubated for 2 h at 37 °C. A secondary antibody anti-rabbit conjugated to HRP  was added and incubated for 1 h at 37 °C. A H2O2 and OPD  1:2000 solution was added and incubated for 5–20 min. The reaction was stopped by adding H2CO4 3M. Absorbance was measured at 492 nm. Serial dilutions of pre-immune and immune serums and purified anti-Ccα6_antibody were tested by duplicate, and wells incubated with BSA 0.5% were used as controls , and transferred onto nitrocellulose membrane. The membrane was blocked for 1 h at room temperature in PBST (0.05% Tween-20 in PBS) containing 1% BSA. Primary antibody incubation was performed with immune serum or purified anti-Ccα6 antibody overnight at 4 °C at 1:100 or 1:50 dilution, respectively. After washing in PBST, membranes were incubated for 1 h at room temperature with a polyclonal goat anti-rabbit HRP-conjugated secondary antibody . Finally, membranes were washed in PBST and developed using the ECL Western Blotting detection reagents   .Adult brains were dissected and fixed with 4% formaldehyde in phosphate-buffered saline for 35 min. Next, samples were washed 3 × 15 min with PBS + 0.1% triton (PBT) and blocked overnight with PBS + 0.1% triton + BSA 5% (PBTX). Then, they were incubated overnight with the primary antibody diluted in PBTX, washed 3 × 15 min in PBT, incubated with secondary antibodies diluted in PBTX for 2 h, mounted with Vectashield mounting medium  with DAPI in microscopy slides, and stored at 4 °C. The primary antibodies used were anti-Ccα6 antibody serum (1:500) and purified anti-Ccα6 antibody (1:50) to recognize the N-terminal region of the α6-subunit of the receptor see  and antiRabbit immunization with a peptide corresponding to the N-terminal domain of the Ccα6 protein produced an immune serum containing the anti-Ccα6 antibody, which was further purified by affinity chromatography. Both immune serum and purified anti- Ccα6 antibody were validated by ELISA assay  and WestD. melanogaster ) or suppression (α6-dsRNA (2)) in the antibody staining  and exon 3b (3b8aCcα6) in D. melanogaster restored susceptibility to spinosad in a α6-deficient strain, indicating that medfly 3b8aCcα6 isoform is enough to mediate spinosad toxicity. Therefore, alternative hypotheses that explain the resistant phenotype conferred by 3aQ68*Ccα6 and 3aAG>ATCcα6 alleles, both encoding 3b8aCcα6 isoform, need to be tested, as for instance the hypothesis that truncated 3a isoforms may interfere with the correct assembly of nAChR receptors in medfly, previously formulated by Ureña et al. [Considering that an alteration in the location of a membrane protein can affect its function, we hypothesize that this could be the reason why alleles t et al.  demonstrD. melanogaster, subunits Dα1, Dα3, Dβ1, Dβ2, Dα7 were mainly localized in the optic lobe, spread in the neuropils, the lamina, the medulla, the lobula, and the lobula plate [Our work also gives some light on the topic of α6 subunit location in the nervous system. Different nAChR subunits were found in the developmental stages of embryo, larva, pupa, and adult of some insect species . In adulla plate ,27,28. Dla plate . Regardila plate . Our resla plate . Further


We used chloroquine (autophagy inhibitor) and reactive oxygen species (ROS) scavenger N-acetylcysteine in conjunction with 1α,25(OH)2D3 to explore the effect on PGCs. The results showed that 10 nM of 1α,25(OH)2D3 increased PGC viability and ROS content. In addition, 1α,25(OH)2D3 induces PGC autophagy according to the gene transcription and protein expression levels of LC3, ATG7, BECN1, and SQSTM1 and promotes the generation of autophagosomes. 1α,25(OH)2D3-induced autophagy affects the synthesis of E2 and P4 in PGCs. We investigated the relationship between ROS and autophagy, and the results showed that 1α,25(OH)2D3-induced ROS promoted PGC autophagy. The ROS-BNIP3-PINK1 pathway was involved in PGC autophagy induced by 1α,25(OH)2D3. In conclusion, this study suggests that 1α,25(OH)2D3 promotes PGC autophagy as a protective mechanism against ROS via the BNIP3/PINK1 pathway.Vitamin D (VD) is one of the important nutrients required by livestock; however, VD deficiency is reported to be widespread. Earlier studies have suggested a potential role for VD in reproduction. Studies on the correlation between VD and sow reproduction are limited. The aim of the current study was aimed to determine the role of 1,25-dihydroxy vitamin D Ovarian granulosa cells (GCs) play a pivotal role in follicle growth and atresia. Autophagy is a process of self-phagocytosis widely present in almost all eukaryotes and is one of the degradation pathways of redundant or abnormal cellular components. The molecular pathways that regulate autophagy are highly conserved. As an important autophagy-related protein, ATG7 is associated with autophagosome formation. BECN1 (Beclin1) forms a complex with the class III phosphoinositol 3-kinase molecule Vps34, which initiates and promotes autophagy. Microtubule-associated protein 1 light-chain 3 (LC3) is essential for the formation and maturation of autophagosomes. SQSTM1P62) protein functions as a selective autophagy receptor for the degradation of substrates. In the ovary, GC autophagy affects follicle development. Autophagy occurs in GCs of porcine follicles  protein . Inadequ3 2D3). Studies conducted over the past 20 years have found that VD plays a vital role in maintaining the regular female reproductive system. Vitamin D and its clinical implications regarding the developmental competence and fertilization of oocytes has prompted researchers’ attention. A recent study revealed that in the case of VD deficiency and hypocalcemia at the same time, a significant reduction in oocyte retrieval after ovarian stimulation was observed, and the generated oocytes showed a poor maturation ability  / [control wells OD − blank wells OD] × 100%. At least three independent replicates were performed.The CCK-8  proliferation assay was used to evaluate the cell viability of PGCs. The cells were seeded at a specific density 2D3, chloroquine (10 μM), and NAC (4 mM) for 24 h. DNA was extracted using Universal Genomic DNA Purification Mini Spin Kit (Beyotime). RNA was extracted using SimplyP Total RNA Extraction Kit  according to the manufacturer’s protocol. Briefly, cells were collected, and 300 μL RIPA was added. Then, the binding solution was added, the mixture was transferred to the purification column and centrifuged at 12,000× g for 30 s, and the liquid in the tube was discarded. After that, washing liquid was added and centrifuged at 12,000× g for 30 s. The RNA purification column was transferred to the RNA eluent tube, and 40 μL eluent was added and centrifuged at the highest speed  for 30 s. The purified RNA was obtained. The process of DNA extraction is similar. Related solvents were added successively and the purified DNA was extracted by centrifugation. The concentration of DNA and RNA was measured by microvolume spectrophotometer . The A260/A280 ratio ranges from 1.9 to 2.0. RNA (500 ng) was reverse transcribed into cDNA according to the FastKing cDNA First Strand Synthesis Kit instructions . Then, the mRNA expression level of genes was measured using ChamQ SYBR qPCR Master Mix  in a StepOnePlus real-time PCR System . The reaction volume was 20 μL: 10 μL 2×ChamQ SYBR qPCR Master Mix, 0.4 μL F-primer (10 μM), 0.4 μL R-primer (10 μM), 5 μL template DNA/cDNA, 4.2 μL ddH2O. The primer sequences are shown in −∆∆Ct method was used to calculate the relative gene expression. All samples were run in triplicate.Approximately 1 × 106 cells/well were seeded into 24-well plates. After being attached, PGCs were treated with 10 nM 1α,25(OH)2D3, chloroquine (10 μM), and NAC (4 mM) for 24 h. Protein was extracted with RIPA buffer (Beyotime) following the manufacturer’s protocol and quantified using the bicinchoninic acid (BCA) protein assay kit (Beyotime). Briefly, after the PGCs were treated, the medium was removed and the cells were washed twice with PBS. RIPA buffer was treated with the cells for 30 min (on ice). The supernatant was collected by centrifugation at 12,000 r/min. The BCA working solution was prepared, the protein standard concentration was 0.5mg/mL, and the protein standard was added to the 96-well plate according to the following amounts: 0, 1, 2, 4, 8, 12, 16, 20 μL. Appropriate volume protein samples were added to standard wells (refill to 20 μL per well). BCA working solution (200 μL) was added to each well for 30 min at 37 °C. The wavelength between A562–595 nm was determined, and the protein concentration was calculated according to the standard curve. The proteins were adjusted to the same concentration by sample buffer. Cytosolic protein (20 μg) was subjected to 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Beyotime) in a Bio-Rad wet Blot Transfer Cell apparatus . The obtained membranes were blocked in QuickBlockTm Western’s blocking buffer (Beyotime) for 1 h at room temperature. Membranes were washed in TBST  and incubated with primary antibodies: β-actin (ACTB)  , LC3  , BNIP3  (Abcam), PINK1  , STAR  , P62   in QuickBlock™ Primary Antibody Dilution Buffer (Beyotime) overnight at 4 ℃. Membranes were then washed and labeled for 2 h at room temperature with anti-rabbit HRP-conjugated IgG goat (1:4000 dilution) or anti-mouse HRP-conjugated IgG (1:4000 dilution)  diluted in QuickBlock™ Secondary Antibody Dilution Buffer (Beyotime). Finally, membranes were washed in TBST, and the protein bands were visualized with a chemical luminous imaging system .Approximately 1 × 106 cells/well were seeded into 24-well plates. After being attached, PGCs were treated with 10 nM 1α,25(OH)2D3 in the presence/absence of chloroquine (10 μM) for 24 h. E2 and P4 in the medium were measured by the specific ELISA kit  following the manufacturer’s protocol. Briefly, medium in the co-culture system was collected by centrifuging at 1000 g for 10 min at 4 °C, and the liquid supernatant was used for steroid assays. Each sample was measured 5 times and averaged. The inter- and intra-assay CVs of E2 were 5.6% and 3.4%, and that of P4 were 6.9% and 7.9%. The minimum detected concentrations of E2 and P4 were 4.8 pg/mL and 1.45 ng/mL, respectively.Approximately 1 × 10ROS generation was detected by DCFH-DA (Beyotime). Fluorescence intensity was measured by a fluorescence microplate reader, fluorescence microscope, or flow cytometry.6 cells/well were seeded into 24-well plates. After being attached, PGCs were treated with 10 nM 1α,25(OH)2D3 in the presence/absence of N-acetylcysteine  for 24 h. After treatments, the cells were co-cultured with 10 μM DCFH-DA for 30 min, the residual DCFH-DA was removed, and the cells were washed with PBS 3 times. The intracellular fluorescence was read by a fluorescence microplate reader at an excitation/emission wavelength of 488/525 nm. Each sample was measured 3 times and averaged.Fluorescence microplate reader: approximately 1 × 106 cells/well were seeded into 24-well plates. After being attached, PGCs were treated with 10 nM 1α,25(OH)2D3 in the presence/absence of N-acetylcysteine (4 mM) for 24 h. Then the cells were stained with DCFH-DA as described above. Cells were observed under a fluorescence microscope, and fluorescence images were obtained. LED intensity, integration time and camera gain were fixed during taking pictures . Image J software was used to process the images, and the mean fluorescence values of different groups were calculated.Fluorescence microscope: approximately 1 × 106 cells/well were seeded into 24-well plates. After being attached, PGCs were treated with 10 nM 1α,25(OH)2D3 for 24 h. The cells were stained with DCFH-DA as described above. After staining, cells in each group were collected (about 1 × 106 cells/mL), and ROS was detected by flow cytometry (BD FACSAria™ III)  within 30 min. The excitation light was 488 nm, and the emission light was 525 nm. Fluorescence was detected by the FL1 channel. Samples were acquired on a flow cytometer using a stop condition of 10,000 events on the gate of interest. Using the flow cytometry software, dot plots of FSC (on the X-axis) and SSC (on the Y-axis) were opened, and a gate was drawn around the cells of interest. In the experiment, untreated normal cells were set as the control group and the gate position was developed according to the two-parameter scatter plot of the control group. Data were analyzed using the FlowJO software.Flow cytometry: approximately 1 × 106 cells/well were seeded into 24-well plates. After the cells were attached, they were treated with 10 nM 1α,25(OH)2D3 for 24 h. Cells were stained with Mito-Tracker Green (Beyotime) according to the manufacturer’s instructions. After removing the cell culture medium, the cells were incubated with prepared Mito-Tracker Green working solution for 30 min at 37 °C. Then the cells were washed with PBS 3 times. The cells were treated with an anti-fluorescence quenched sealing solution and then observed under a fluorescence microscope (Olympus Corporation). LED intensity, integration time, and camera gain were fixed during picture-taking. Image J software was used to process the images, and the mean fluorescence values of different groups were calculated.Approximately 1 × 106 cells/well were seeded into 24-well plates. After the cells were attached, they were treated with 10 nM 1α,25(OH)2D3 for 24 h. Then, the cells were treated with a mitochondrial membrane potential detection kit (JC-1) (Solarbio) following the manufacturer’s protocol. Briefly, JC-1 staining working solution was added to each well, thoroughly mixed, and then the cells were incubated for 20 min at 37 °C in a cell incubator. The cells were washed 2–3 times with PBS. After staining, cells in each group were collected (about 1 × 106 cells/mL), and the JC-1 signal was visualized by flow cytometry  (BD FACSAria™ III) within 30 min. Green fluorescence was detected through FL1 channel, and red fluorescence was detected through FL2 channel. Samples were acquired on a flow cytometer using a stop condition of 10,000 events on the gate of interest. Using the flow cytometry software, dot plots of FSC (on the X-axis) and SSC (on the Y-axis) were opened, and a gate was drawn around the cells of interest. In the experiment, untreated normal cells were set as the control group, and the gate position was developed according to the two-parameter scatter plot of the control group. Data were analyzed using the FlowJO software.Approximately 1 × 106 cells/well were seeded in 24-well plates. After being attached, PGCs were treated with 10 nM 1α,25(OH)2D3, chloroquine (10 μM), and NAC (4 mM) for 24 h. MDC (monodansylcadaverine) was used as a tracer of autophagic vesicles. The autophagosomes are marked as clear green dots under the fluorescence microscope. After treatment, the cells were treated with MDC (0.05 mM)  and DAPI (1 μg/mL)  (Solarbio) following the manufacturer’s protocol. Briefly, the cells were grown with MDC and DAPI at 37 °C for 15 min and fixed immediately with paraformaldehyde (4%) in PBS for 20 min, then observed under a fluorescence microscope (Olympus Corporation). LED intensity, integration time, and camera gain were fixed while taking pictures. Image J software was used to process the images, a total of 200 cells in each sample were analyzed, and the percentage of cells with green spots indicates the percentage of autophagy.Approximately 1 × 106 cells/well were seeded in 24-well plates. After being attached, PGCs were treated with 10 nM 1α,25(OH)2D3 for 24 h. The activity of intracellular SOD and GPX were measured in PGCs using kits  for scientific research following the manufacturer’s protocol. Briefly, after the cells were washed twice with PBS, the cells were carefully scraped off with a cell scraper, and the cell mixture was centrifuged at 1000× g for 10 min, and then the supernatant was discarded. Protein concentration was determined with the BCA assay kit (Beyotime). The results were detected by a visible spectrophotometer (550 nm wavelength) (Thermo Fisher Scientific). Each sample was measured 5 times, and the average of the results was taken. The inter- and intra- assay CVs averaged 7.8% and 6.5%, respectively.Approximately 1 × 10t-tests were used to evaluate the significance of the results between groups. Statistical significance was determined by ANOVA followed by post hoc tests. The Tukey–Kramer HSD test was used to analyze the differences between the means . p-values < 0.05 were considered statistically significant. All data are presented as the mean ± SM of 3 or more repeated observations from at least 3 independent experiments.Independent 2D3 induces mitophagy through the ROS-BNIP3-PINK1 signaling pathway, which promotes the proliferation and maintains the function of PGCs. Our results provide important information for determining the role of 1α,25(OH)2D3 during ovarian follicular development.In summary, these results demonstrate for the first time that 1α,25(OH)