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  • br Animals Female Sprague Dawley SD rats


    Animals Female Sprague Dawley (SD) rats, aged 3 months with body weight between 180-210 g were housed in cages and acclimatized to standardized lab conditions (temperature 23 ± 2°C, humidity 50 ± 5% and 12 h light-dark cycles). The rats were fed with standard rodent food pellet (Harlan Diet, Rossdoff, Germany) and had free access to tap water ad libitum. All experimental procedures were approved by Institutional Animal Care and Use Ethics Committee, University of Malaya.
    Ovariectomy, drug treatment, vaginal pH probing and vaginal tissue harvesting Bilateral ovariectomy was performed to eliminate the effect of endogenous sex-steroids, following the procedures as previously described (Shahzad et al. 2014). Three weeks after ovariectomy, treatment was commenced and rats (n = 6 per group) were divided into the following groups:
    Quantification of RNA by using Real-time PCR (qPCR) One step Real Time PCR was performed to evaluate gene topotecan with application of TaqMan®RNA-to-CT 2-Step kit, following the method as previously described (Loh et al. 2017). TaqMan® primers were used and were pre-designed (Applied Biosystems, Foster City, CA, USA). The primers used were as follows: CAII (Rn00570700_m1), CAIII (Rn00695939_m1), CAIX (Rn01764733_m1), CAXII (Rn01418250_m1), CAXIII (Rn01493656_g1), V-ATPase A1 (Rn01763276_m1), V-ATPase B1/2 (Rn01765558_m1), AC II (Rn00578713_m1), Na+/K+-ATPase (Rn01533986_m1), GAPDH (Rn0177576_g) and Hprt (Rn01527840_m1). All experiments were carried out in triplicates and data were analysed according to comparative Ct (2−ΔΔCt) method. The relative quantity of target in each sample was determined by comparing the normalized target quantity of the sample to average normalized quantity of the references. Then the amplified region of cDNA was probed by using fluorescence-labelled probe. Validation of the assay was performed in silico by using whole rat genome and in-vitro by using whole rat cDNA (Applied Biosystems, Foster City, CA, USA).
    Immunoperoxidase staining to visualize protein distribution Histology and immunofluorescence were performed following the methods as previously described (Giribabu et al., 2017). In brief, sections were blocked with appropriate normal serum (Santa Cruz, CA, USA) prior to incubation with V-ATase A1 (sc-28801), V-ATPase B1/2 (sc-20943), CAII (sc-17244), CAIII (sc-50715), CAIX (sc-25600), CAXII (sc-25601) and CAXIII (sc-67334) at a dilution of 1:100 in PBS with 1.5% normal blocking serum at room temperature for 1 h. After three times rinsing with PBS, sections were incubated with IgG–fluorochrome-conjugated secondary antibody (Santa Cruz, CA, USA), at a dilution of 1:250 in PBS with 1.5% normal blocking serum at room temperature for 45 min. The slides were rinsed three times with PBS and were mounted with Ultra Cruz mounting medium (Santa Cruz, CA, USA), counterstained with DAPI to visualize the nuclei.
    Western blotting for quantification of protein expression level Western blotting was performed according to the methods as previously described (Shahzad et al. 2017). Once proteins were transferred, membranes were exposed to the same primary antibodies as above. β-actin primary antibody was used as loading control. Following incubation with the primary antibodies, membranes were then incubated with appropriate horseradish peroxidase (HRP) conjugated secondary antibodies.
    Statistical analysis
    Discussion The findings from this study indicated that treatment of sex-steroid deficient ovariectomized rats with MPE restored the vaginal fluid acidity most probably due to an increase in the generation of H+ as reflected by increased levels of CA isoenzymes as well as could be due to an increased extrusion of H+ as reflected by increased levels of V-ATPase isoforms in the vagina. This study showed that MPE extract contains gallic acid, rutin, myricetin, quercetin and kaempferol, with gallic acid was present in the highest amount. Due to its quantity, gallic acid could be responsible for the observed pharmacological effects. However, the effect of other compounds could not be ruled out. As no previous studies revealed the trophic effect of these compounds on the vagina, our study topotecan could be the first to identify their effects.