OBJECTIVEThe determination method of histatins 5 in human saliva with reversed-phase high performance liquid chromatography (HPLC) was developed.

METHODSSalivary samples were collected and diluted with phosphate buffer (pH 2.5). The upper solution was determined with HPLC. Phosphate buffer (pH 3.5) of the mobile phase and C18 column was used throughout the experiment. The detection wavelength was 276 nm.

RESULTSThe linear ranges were 1.0-50.0 microg x mL(-1). The detection limit was 0.12 microg x mL(-1). The relative standard derivations (RSD) of standard solution for reserved time and peak area were 0.68% and 4.13% respectively. The proposed method was applied for analysis of salivary samples and the satisfactory results were obtained. RSD for sample determination was 4.41% and the average recoveries were 88.4%-109.0%.

CONCLUSIONThe method was quick, simple and accurate. Analytical time was less than 15 min. It was adapted for analysis of salivary histatins 5 in salivary samples.

Chromatography, High Pressure Liquid ; Histatins ; Humans ; Saliva

Human salivary histatin 1 (Hst1) exhibits a series of cell-activating properties, such as promoting cell spreading, migration, and metabolic activity. We recently have shown that fluorescently labeled Hst1 (F-Hst1) targets and activates mitochondria, presenting an important molecular mechanism. However, its regulating signaling pathways remain to be elucidated. We investigated the influence of specific inhibitors of G protein-coupled receptors (GPCR), endocytosis pathways, extracellular signal-regulated kinases 1/2 (ERK1/2) signaling, p38 signaling, mitochondrial respiration and Na+/K+-ATPase activity on the uptake, mitochondria-targeting and -activating properties of F-Hst1. We performed a siRNA knockdown (KD) to assess the effect of Sigma-2 receptor (S2R) /Transmembrane Protein 97 (TMEM97)-a recently identified target protein of Hst1. We also adopted live cell imaging to monitor the whole intracellular trafficking process of F-Hst1. Our results showed that the inhibition of cellular respiration hindered the internalization of F-Hst1. The inhibitors of GPCR, ERK1/2, phagocytosis, and clathrin-mediated endocytosis (CME) as well as siRNA KD of S2R/TMEM97 significantly reduced the uptake, which was accompanied by the nullification of the promoting effect of F-Hst1 on cell metabolic activity. Only the inhibitor of CME and KD of S2R/TMEM97 significantly compromised the mitochondria-targeting of Hst1. We further showed the intracellular trafficking and targeting process of F-Hst1, in which early endosome plays an important role. Overall, phagocytosis, CME, GPCR, ERK signaling, and S2R/TMEM97 are involved in the internalization of Hst1, while only CME and S2R/TMEM97 are critical for its subcellular targeting. The inhibition of either internalization or mitochondria-targeting of Hst1 could significantly compromise its mitochondria-activating property.
Endocytosis/physiology* ; Histatins/pharmacology* ; Humans ; Membrane Proteins ; Mitochondria/metabolism* ; RNA, Small Interfering/pharmacology* ; Receptors, G-Protein-Coupled/metabolism* ; Receptors, sigma

OBJECTIVETo study the influence of histatin 1 (Hst1) on the proliferation and migration of human epidermal cell line HaCaT.

METHODS(1) HaCaT cells were routinely cultured and divided into control group, 100, 30, and 3 µg/mL Hst1 groups, 10 ng/mL recombinant human epidermal growth factor (rhEGF) group, and 30 µg/mL Hst1 + 10 ng/mL rhEGF group, according to the random number table (the same dividing method used for following grouping), with 27 samples in each group. NO stimulating factor was added in control group, while Hst1 and(or) rhEGF in corresponding concentration(s) was (were) added in the latter 5 groups. Cell proliferation was assayed by cell counting method at post culture hour (PCH) 24, 48, and 72. (2) HaCaT cells were divided into control group and 100, 30, and 3 µg/mL Hst1 groups, with 27 samples in each group. NO stimulating factor was added in control group, while Hst1 in corresponding concentration was added in the latter 3 groups. Cell cycle was assayed with flow cytometry at PCH 24, 48, and 72, and PI was calculated. (3) HaCaT cells were divided into control group, 30 µg/mL Hst1 group, 10 ng/mL rhEGF group, 30 µg/mL Hst1 + 10 ng/mL rhEGF group, 15 µg/mL Hst1 + 5 ng/mL rhEGF group, and 15 µg/mL Hst1 + 10 ng/mL rhEGF group, with 10 samples in each group. NO stimulating factor was added in control group, while Hst1 and(or) rhEGF in corresponding concentration(s) was (were) added in the latter 5 groups. Cells in each group were divided into two portions: cells in one portion were treated by mitomycin C for 2 hours, while cells in the other portion were not. Scratching assay was conducted in both portions of cells. Cell migration was measured at post scratching hour (PSH) 0, 16, and 24, and the wound-area healing rate was calculated. Data were processed with analysis of variance, and LSD- t test or Dunnett t test was applied in paired comparison among groups.

RESULTS(1) At PCH 24, the cell numbers in 10 ng/mL rhEGF group and 30 µg/mL Hst1 + 10 ng/mL rhEGF group were significantly higher than that in control group (with t values respectively 3.813, 5.410, P < 0.05 or P < 0.01). Except for cell numbers in 30 µg/mL Hst1 group and 3 µg/mL Hst1 group at PCH 48, cell numbers in the other groups as treated by Hst1 and (or) rhEGF were significantly higher than those in control group at PCH 48 and 72 (with t values from 7.754 to 24.979, P values all below 0.01). At PCH 72, the cell number was obviously higher in 100 µg/mL Hst1 group [(19.21 ± 0.59)×10⁴] than in 30 µg/mL Hst1 group [(16.19 ± 0.53)×10⁴)] and 3 µg/mL Hst1 group [(15.38 ± 0.13)×10⁴], with t values respectively 11.391, 19.017, P values all below 0.01. The cell number was higher in 30 µg/mL Hst1 + 10 ng/mL rhEGF group than in 30 µg/mL Hst1 group, 3 µg/mL Hst1 group, and 10 ng/mL rhEGF group (with t values from 4.579 to 34.884, P < 0.05 or P < 0.01). Cell numbers in all groups increased with prolongation of time. (2) Compared with those in control group at PCH 24 and 48, the percentage of cells in G0/G1 phase was decreased, the percentage of cells in S phase was increased (except for cell percentage of 30 µg/mL Hst1 group at PCH 24), and PI value was significantly increased in 100 µg/mL Hst1 group and 30 µg/mL Hst1 group (with t values from 4.752 to 16.104, P values all below 0.01). The PI value in 3 µg/mL Hst1 group was obviously higher than that in control group only at PCH 48 (t = 4.609, P < 0.01). At PCH 72, only the PI value in 100 µg/mL Hst1 group was higher than that in control group (t = 8.005, P < 0.01). Compared among the groups treated by Hst1, the percentage of cells in G0/G1 phase showed an elevating trend, and the percentage of cells in S phase and the PI value showed a declining trend along with the decrease in Hst1 concentration at each time point. Compared within each group treated by Hst1, the percentage of cells in G0/G1 phase declined first and then elevated, while the percentage of cells in S phase and the PI value elevated first and then declined along with prolongation of time. (3) Without treatment of mitomycin C, the wound-area healing rate in 30 µg/mL Hst1 group (75.9 ± 3.9)% at PSH 16 was significantly higher than those in control group and 10 ng/mL rhEGF group [(53.0 ± 3.5)%, (61.7 ± 2.5)%, with t values respectively 12.241, 7.598, P values all below 0.01], but lower than those in 30 µg/mL Hst1 + 10 ng/mL rhEGF group, 15 µg/mL Hst1 + 5 ng/mL rhEGF group, and 15 µg/mL Hst1 + 10 ng/mL rhEGF group [(95.0 ± 4.1)%, (97.0 ± 3.7)%, (80.5 ± 5.9)%, with t values from -11.324 to -2.502, P < 0.05 or P < 0.01]. After being treated by mitomycin C, the wound-area healing rate in 30 µg/mL Hst1 group at PSH 16 [(54.1 ± 4.5)%] was higher than that in control group [(35.8 ± 5.7)%, t = 7.790, P < 0.01], but lower than that in the same Hst1 concentration but without mitomycin C treatment group (t = -10.863, P < 0.01). There was no statistically significant difference in the wound-area healing rate between 30 µg/mL Hst1 group and other groups treated by Hst1 and rhEGF at PSH 16 (with t values from 0.061 to 2.030, P values all above 0.05). Compared within each group with or without treatment of mitomycin C, the wound-area healing rate at PSH 16 was not significantly different from that at PSH 24 (with F values from 0.856 to 3.062, P values all above 0.05).

CONCLUSIONSHst1 can promote the proliferation and migration of HaCaT cells. It has synergic effect with rhEGF on the promotion of cell proliferation, but their synergic effect on cell migration is not obvious.

Cell Cycle ; drug effects ; Cell Line ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Epidermis ; cytology ; Histatins ; pharmacology ; Humans ; Wound Healing