TT-10 may attenuate ibuprofen-induced ovarian injury in mice by activating COX2-PGE2 and inhibiting Hippo pathway.

Ibuprofen (IBU) is a non-steroidal anti-inflammatory drug that has been found in recent years to cause ovarian damage. The aim of this study is to explore the molecular mechanisms of IBU damage to the ovary and drugs to combat it. We established in vivo (IBU doses of 50, 100 and 200 mg/kg-day) and in vitro (IBU concentrations of 50, 100 and 200 µM in culture medium) models of ovarian damage in mice simulating clinical doses and found that IBU not only caused ovarian damage in mice in a dose-response relationship, but also decreased estradiol (E2) and prostaglandin E2 (PGE2) levels in serum/media with increasing IBU doses. In damaged ovaries, the cyclooxygenase 2 (COX2)-PGE2 pathway is inhibited, the Hippo pathway is activated, circPVT1 is decreased, and miR-149 is elevated. TT-10 is an activator of YES-associated protein (YAP)-transcriptional enhancer factor domain activity. Then, 100 µM IBU-induced ovarian damage model was selected for YAP activation (Hippo pathway inhibition) experiment, and TT-10 was found to interfere with IBU-induced ovarian damage and increase E2 level in the medium, and 10 µM of TT-10 had the best protective effect. TT-10 also inhibited the Hippo pathway, activated the COX2-PGE2 pathway, elevated circPVT1 expression, and decreased miR-149 expression in the ovary. It has been hypothesized that clinical doses of IBU damage mouse ovaries by inhibiting COX2-PGE2 and activating the Hippo pathway, whereas TT-10 protects the ovaries through the inverse regulation of these two pathways.

Reduction of excessive unfolded protein response by 4-phenylbutyric acid may mitigate procymidone-induced testicular damage in mice by changing the levels of circRNA Scar and circZc3h4.

Procymidone (PCM) exposure below the no-observed-effect level triggers changes in circRNA Scar and circZc3h4 and overactivation of the unfolded protein response (UPR) in mice, culminating in testicular injury. The 4-phenyl butyric acid (4-PBA) is known to stabilize proteins and reduce the UPR. This study employed an in vitro system in which mouse testes were cultured with 1 × 10-5 M PCM and varying concentrations (0, 20, 40, and 80 mM) of 4-PBA; 4-week-old male mice were subsequently treated with 100 mg/kg/d PCM (suspended in corn oil) and/or 100 mg/kg/d 4-PBA for 21 d, consecutively. The treatments were as follows: the negative control (NC) group was orally administered corn oil; the positive control (PC) group was orally administered PCM; the 4-PBA group was intraperitoneally injected with 4-PBA; the 4-PBA-I group was orally administered PCM and 4-PBA simultaneously; the 4-PBA-II group received daily administration of 4-PBA 24 h prior to PCM; and the 4-PBA-III group was intraperitoneally injected with 4-PBA for 7 d after 21 d of PCM administration. However, the 4-PBA intervention groups showed no considerable changes in the overall or testicular appearance of mice. In vitro, 4-PBA inhibited the PCM-induced testicular injury, with the most significant effect observed at 80 mM. In vivo, the 4-PBA-III group exhibited the best in vivo effects. Our findings indicate that 4-PBA conferred testicular protection by decreasing PCM-induced circRNA Scar, elevating circZc3h4, and suppressing UPR both in vitro and in vivo. It has been hypothesized that 4-PBA mitigates testicular damage by reducing excessive UPR levels.

[Change of the retinoic acid pathway in hypothalamus and pituitary damage induced by combined exposure to low-level Pb~(2+) and 1-nitropyrene in mice].

OBJECTIVE: To observe the expression of the retinoic acid(RA) pathway in hypothalamus and pituitary damage induced by combined exposure of low-level lead and 1-nitropyrene in mice, and to explore the relationship between the changes of RA pathway and hypothalamus and pituitary damage. METHODS: A total of 84 4-week-old ICR mice were randomly divided into the control group, Pb~(2+) tainted group(0.008 mg/L), 1-NP tainted group(0.1 mg/kg), low(0.008 mg/L Pb~(2+)+0.004 mg/kg 1-NP), medium(0.008 mg/L Pb~(2+)+0.02 mg/kg 1-NP), and high-dose co-toxicity group(0.008 mg/L Pb~(2+)+0.1 mg/kg 1-NP) according to body weight, with 14 mice in each group. Among them, Pb~(2+) was provided by lead acetate, added to deionized water and ingested by mice drinking freely, 1-NP was given by intraperitoneal injection, 1-NP was administered by intraperitoneal injection. Record daily water intake and food intake. After 21 consecutive days of exposure, body mass was measured, histological changes in the hypothalamus and pituitary were observed under an optical microscope, and lead content in brain tissue was measured by atomic absorption spectrometry. The real-time fluorescence quantitative PCR was used to detect the abundance of retinoic acid pathway members and c-Jun N-terminal kinases genes(Jnks), and the western blot method was used to detect expression levels of acetaldehyde dehydrogenase 2(ALDH2), cytochrome P450 family member 26A1(CYP26a1) proteins. RESULTS: There was no difference in the mean weekly water intake and food intake of the mice in each group. The body weight of the high-dose co-toxicity group mice((27.4±1.9)g) was lower than that of the control group((29.8±2.3)g)(P<0.05). The level of serum follicle-stimulating hormone(FSH) in the middle and high dose co-toxicity groups((265.01±2.99), (260.42±3.61)pg/mL, respectively) was lower than that in the control group((279.00±1.30)pg/mL, P<0.05). The content of Pb~(2+) in the brain of each group containing Pb~(2+) was higher than that of the control group. In the hypothalamic and pituitary tissues, the abundance of Adh1, Adh2, Rar and Rxr, and ALDH2 levels in the medium and high dose co-toxicity groups were higher than those in the control group(P<0.05). Cyp26a1 gene abundance and protein levels were lower in the medium and high dose co-toxicity groups than in the control group(P<0.05). The abundance of Jnks in the high-dose co-toxicity group was higher than that in the control group(P<0.05). CONCLUSION: Continuous exposure to 0.008 mg/L Pb~(2+)+0.1 mg/kg 1-NP for 21 days can cause damage to the hypothalamus and pituitary of mice, and activate the RA signaling pathway.

4-Phenylbutyric acid may prevent mouse ovarian and uterine damage due to procymidone-induced alteration of circRNA Scar and circZc3h4 levels by controlling excessive unfolded protein response.

Procymidone (PCM) below the no-observed-adverse-effect-level (NOAEL) has previously been proven to induce ovarian and uterine damage in adolescent mice due to its raised circRNA Scar, decreased circZc3h4, and overactivated unfolded protein response (UPR). Also, 4-phenylbutyric acid (4-PBA) inhibits histone deacetylase and endoplasmic reticulum stress, reduces UPR, improves metabolism, and ensures homeostasis within the endoplasmic reticulum. In this study, 20, 40 and 80 mM of 4-PBA were utilized respectively to intervene the damage caused by 1.0 × 10-5 M PCM to ovaries and uterus in vitro culture. Besides, 100 mg/kg /d 4-PBA was intraperitoneally injected to female adolescent mice before, during and after oral administration of 100 mg/kg /d PCM for prevention and cure to observe tissue changes in the ovaries and uteri, and levels of circRNA Scar, circZc3h4 and UPR members. Our findings demonstrated that in vitro experiments, all doses of 4-PBA could inhibit ovarian and uterine damage caused by PCM, and the effect of 80 mM was especially noticeable. In the in vivo experiments, the best results were obtained when PCM was given with simultaneous 4-PBA intervention, i.e., minimal ovarian and uterine damage. Both in vivo and in vitro, 4-PBA in the ovary and uterus resulted in decreased circRNA Scar levels, increased circZc3h4 abundance, and moderately elevated levels of UPR members. So, it is suggested that 4-PBA moderately activates UPR, partially or completely antagonizing the elevated circRNA Scar and decreased circZc3h4 and consequently preventing PCM-induced ovarian and uterine damage effectively in adolescent mice.

TT-10 may elevate YAP and repair mouse uterine damage resulting from the inhibition effect of ibuprofen on COX2-PGE2 and YAP.

Ibuprofen (IBU) is an emerging environmental contaminant that, in high doses, can damage reproductive organs in humans and other mammals. Recently, its effects on the uterus have been investigated. It is known that the COX2-PGE2 pathway and Yes-associated protein (YAP) are involved in female reproductive organ development and form a COX2-PGE2-EP2-Gas-ß-catenin-YAP-COX2 positive feedback loop, in addition, TT-10, a pharmacological product, has been found to increase YAP. In this study, IBU was orally administrated to female mice for 7 d at doses of 0, 50, 100, and 200 mg/kg·bw/day (control, low, medium, and high doses, respectively). In addition, 0, 50, 100, and 200 µmol/L IBU was added in vitro to cultured uterine cells for 7 d at control, low, medium, and high doses, respectively; then, 0, 5, 10, and 20 µmol/L TT-10 were given to the in vitro uterine culture containing 100 µmol/L IBU to observe the effect of YAP activation. The results showed that medium and high doses of IBU inhibited the COX2-PGE2 pathway, decreasing YAP and increasing pYAP, leading to reduced circPVT1, elevated miR-149, and increased apoptosis, ultimately damaging the uterus. Conversely, 10 µmol/L TT-10 maximally enhanced YAP, which regulated COX2-PGE2 pathway activation, increased circPVT1, and decreased miR-149, and promoted cell proliferation, preventing uterine damage. This suggests that IBU may cause uterine damage by inhibiting the COX2-PGE2 pathway and YAP, and that appropriate doses of TT-10 may repair this damage by elevating YAP and stimulating COX2 via the feedback loop.

Accentuated Hippo pathway and elevated miR-132 and miR-195a lead to changes of uteri and ovaries in offspring mice following prenatal exposure to vinclozolin.

Vinclozolin (VCZ) has been identified as a broad-spectrum fungicide and an environmental endocrine disruptor. Also, the Hippo signaling pathway controls organ size by regulating cell proliferation and apoptosis, and moreover, overexpression of microRNA-132 (miR-132) and microRNA-195 (miR-195) inhibits cell proliferation and promotes apoptosis. So, in this study, the experimental mice were orally given 400 mg/kg/day VCZ (suspended in corn oil) at gestational day 12-18, while those of the control group were fed with corn oil of equal volume. Then unilateral ovaries and mid-uteri were isolated from 10 randomly-selected mice at the postnatal 1st week (7 days), 3rd week (20-21 days), and 7th week (48-49 days) respectively to observe gene levels, while 6 of the contralateral ovaries and uteri were subsequently examined for proteins respectively. Besides, 16 from both groups were determined with serum estradiol (E2) at week 7, of which 6 were randomized for histological observation. Here we found the levels of E2 reduced in VCZ-group at week 7, with fewer follicles and injured endometrium. Meanwhile, in VCZ mice of all ages, increased miR-132 and miR-195a, decreased G protein-coupled estrogen receptor (GPER), elevated phosphorylated large tumor suppressor (pLATS) and phosphorylated yes-associated protein (pYAP), and decreased yes-associated protein (YAP) were observed in their ovaries and uteri. These findings suggested ovarian and uterine dysplasia in the offspring induced by gestational VCZ-exposure were mainly attributed to higher miR-132 and miR-195a and accentuated Hippo-pathway.

The joint action of unfolded protein response, circZc3h4, and circRNA Scar in procymidone-induced testicular injury in adolescent mice.

Procymidone (PCM) is a low toxicity fungicide, and an endocrine-disrupting chemical (EDC) that particularly damages the reproductive system of male vertebrates. In present study, adolescent mice in control, low-, medium-, and high-dose groups were orally administered 0 (equal volume of soybean oil), 50, 100, and 200 mg/kg/day PCM, respectively, for 21 days. Additionally, a three-dimensional culture of mouse testes was performed in vitro, and the control, low dose (0.33 × 10-5  M), medium dose (1 × 10-5  M), and high dose (3 × 10-5  M) PCM groups were established. We have found that, under both in vivo and in vitro conditions, all doses of PCM caused damage to mouse testes. Moreover, the levels of circZc3h4 RNA and Zc3h4 decreased while miR-212 increased in all treatment groups, with a corresponding rise in circRNA Scar and fall in Atp5b, compared to those in the control group, and all the changes showed a dose-response relationship. Besides, we have identified that low doses of PCM could activate the Ire1-Xbp1 pathway, whereas the medium and high doses activated the Perk-Elf2α-Atf4, Ire1-Xbp1, and Atf6 pathways. And it is, therefore, speculated that the unfolded protein response (UPR), circZc3h4 and circRNA Scar may have taken joint action in testicular injury in adolescent mice induced by PCM at the no observed adverse effect level (NOAEL, 100 mg/kg/day) and below NOAEL doses.

Combined effect of unfolded protein response and circZc3h4, circRNA Scar in mouse ovary and uterus damage induced by procymidone.

Procymidone (PCM) is a fungicide commonly used to prevent and control plant diseases, and it is also an environmental endocrine disruptor that has a typical anti-androgen effect on the function and/or structure of the vertebrate reproductive system. The activation of the unfolded protein response (UPR) will fold the protein correctly to ensure the cell's survival. PCM regulates GRP78 by affecting the level of hormones, and there is a regulatory relationship between the UPR, the circRNAs and the miRNAs. In vivo experiments, PCM (suspended in soybean oil) was orally administered to adolescent female mice for 21 days in 3 different doses of 50 mg kg-1 day-1 (low dose), 100 mg kg-1 day-1 (medium dose) and 200 mg kg-1 day-1 (high dose) to cause ovaries and uteruses damage, and in vitro experiments, various doses of PCM from 0.33 × 10-5 (low dose) to 1 × 10-5 (medium dose) then 3 × 10-5 M (high dose) were used to induce injury on the ovaries and uteri of the mice. We found out that both in vivo and in vitro, PCM caused dose-dependent damages to the ovaries and uteri, increased their circRNA Scar levels and decreased circZc3h4 abundance. Also, all UPR signaling pathways in the low-dose group and some in the middle-dose group were activated. It is speculated that UPR may antagonize the partial ovarian and uterine damage in adolescent mice induced by PCM at doses less than NOAEL via changes in circZc3h4 and circRNA Scar.

[Expression of retinoic acid signaling pathway in uterus injury induced by procymidone in adolescent mice].

OBJECTIVE: To investigate the expression of key genes and proteins of retinoic acid signaling pathway in procymidone-induced uterine injury in adolescent mice, and analyze the relationship between the signaling pathway and female reproductive damage. METHODS: The 3-week age ICR mice were randomly divided into low, medium, and high-dose groups and one control group with 8 mice in each group by weight. The low, medium and high dose groups were respectively given 50, 100 and 200 mg/(kg·d) procymidone orally for 21 days continuously, while the control group was given equal volume of soybean oil. After the mice were sacrificed, the uterus was taken from both sides for observing the histological changes in the cross-sectional slices of the uterus, the detection of the expression abundance of genes which related to the retinoic acid signaling pathway by the real-time fluorescent quantitative PCR, and the measurement of ALDH2 and CYP26 a1 proteins expression by Western blot. RESULTS: The body weight of mice in low-dose, medium-dose and high-dose groups were(27.50±1.49) g, (27.72±1.40) g and(26.89±1.19) g, respectively, which were lower than those in control group(31.48±1.14) g(P<0.05). The density of uterine lining monolayer columnar epithelium and lamina propria tubular uterine glands gradually decreases, at the same time the uterine folds become less with the dose of procymidone increases. adh1, ad/2, aldh1a1 in each experimental group were higher than those in the control group(P<0.05); the expression levels of aldh1a2 and aldh1a3 genes in the middle and high dose groups were higher than those in the control group(P<0.05); the expression levels of retinoic acid nuclear receptor rarα, rarγ, rxrα and rxrß genes in the high-dose group were higher than those in the control(P<0.05); yet the expression levels of cyp26a2 and cyp26a3 in the high-dose group were lower than those in the control group(P<0.05); the jnk family in medium and high dose groups were higher than the control(P<0.05). The expression of ALDH2 in each experimental group was higher than that in the control group, and increased with the increase of the dose(P<0.05); the expression of CYP26 a1 in each experimental group was not significantly different from that of the control group. CONCLUSION: The retinoic acid signal pathway is activated in procymidone-induced uterine injury in mice, then regulates the increase of the expression of jnk family, leading to the damage.

SBS Modified Bitumen with Organic Layered Double Hydroxides: Compatibility and Aging Effects on Rheological Properties.

SBS-modified bitumen (SMB) is susceptible to aging, which seriously influences its service performance and life. In order to strengthen the anti-aging ability of SMB, triethoxyvinylsilane was designed to organically modify layered double hydroxides (LDHs) and was applied to modify SMB. The dispersibility and storage stability of LDHs in SMB were markedly enhanced after triethoxyvinylsilane organic modification, and the compatibility and storage stability of SBS in bitumen were simultaneously enhanced. Compared with SMB, the introduction of LDHs and organic LDHs (OLDHs) could ameliorate the high-temperature properties of SMB, and the thermostability of SBS in bitumen at a high temperature was also distinctly improved, especially OLDHs. After aging, due to the oxidation of molecular bitumen and the degradation of molecular SBS, SMB became hardened and brittle, and the rheological properties were significantly deteriorated, which had serious impacts on the performance of SMB. LDHs can mitigate the detriment of aging to bitumen and SBS, and the deterioration of the rheological properties of SMB is obviously alleviated. As a result of the better dispersibility and storage stability, OLDHs exerted superior reinforcement of the anti-aging ability of SMB.

Vinclozolin-induced mouse penile malformation and "small testis" via miR132, miR195a together with the Hippo signaling pathway.

Vinclozolin (VCZ) is a fungicide with antiandrogen activity. Exposure to VCZ in maternal uterus may cause uterine, ovarian and testicular damage, hypospadias and prostate abnormality in the offspring. Hippo pathway, which is highly conservative and may be activated by miR132 and miR195a, can control organ size and tissue regeneration, and participate in injury and deformity. In the present study, VCZ was found to have caused penile malformation in the male offspring and also induced "small testis" when it was administered to the pregnant mice orally at a dose of 400 mg kg-1 day-1 on Days 12-18 of gestation. At 1, 3 and 7 weeks of age, VCZ could increase miR132, Mst1, Sav1, phosphorylated Yes-associated protein (pYap) and pLats, and decrease Yap in offspring penises and testes. Besides, it could also raise miR195a both in the testes of 1, 7-week and in the penises of all the three ages. In addition, we found the levels of some cyclin (Ccn) genes elevated in the testes, the expression of the androgen receptor (Ar) gene dereased and Jnks changed in the penises of offspring aged 1, 3 and 7 weeks. The results suggest that that gestational VCZ exposure could not only increase miR132 and miR195a in penises and testes of the offspring, but also activate Hippo pathway and down-regulate Ar. These may directly inhibit cell proliferation, accelerate cell death by up-regulating the expression of some Ccns, and ultimately lead to penile and testicular damage and malformations in the offspring.

Flutamide induces uterus and ovary damage in the mouse via apoptosis and excessive autophagy of cells following triggering of the unfolded protein response.

Intrauterine exposure to flutamide not only causes abnormal development of the reproductive organs in male offspring, but also damages ovaries and uteri. The unfolded protein response (UPR) is believed to play an important role in embryo development and teratogenic processes. In the present study, pregnant mice were administered either flutamide (300mg kg-1 day-1, p.o.) on an equivalent volume of soybean oil (control) on Days 12-18 of gestation. Eight weeks after birth, female offspring in the flutamide-treated group had a lower bodyweight and lower ovarian and uterine weights, but there was no significant difference in uterine and ovarian weights normalised by bodyweight between the flutamide-treated and control groups. Furthermore, histopathological changes were observed in all uteri and ovaries in the flutamide-treated group, with fewer and less-developed follicles in the ovaries. In both the uteri and ovaries, flutamide increased the expression of UPR members, although the expression of cell cycle-related genes remained unchanged compared with the control group. Flutamide increased the expression of all autophagy- and apoptosis-related genes evaluated in the uterus, as well as some in the ovary. The results suggest that the in utero exposure of mice to flutamide may contribute to uterine and ovarian damage in the offspring, with endoplasmic reticulum stress possibly triggered by the UPR leading to the induction of excessive autophagy and apoptosis.

[Injury of procymidone on testis and sperm in the male adolescent mice].

OBJECTIVE: To investigate the damage of procymidone to penile, testis and sperm in adolescent male mice. METHODS: 4-week-old male ICR mice were allocated randomly to the treatment groups and the control group, with 8 mice in each group. Procymidone was administered to mice by gavage with dose 50 mg/kg(low dose), 100 mg/kg and 200 mg/kg, while the control group was given only an equal volume of soybean oil. On the 11 th days, the mice were sacrificed by spinal dislocation, their body masses were weighed, and the anogenital distance(AGD), testicular volume and penis length were measured. Furthermore the weight of the testes, epididymis and penis were weighed, the organ coefficients were calculated, and then a testis on one side and 1/2 penis tissue were fixed in 4% paraformaldehyde and used for histology analysis. The testis on the other side and the remaining 1/2 penis were used to detect androgen content and Caspase-3, Caspase-9, Caspase-12 and Bax gene expression. At the same time, one epididymis was randomly selected to detect sperm motility, density and morphology. RESULTS: The weight, penis length and testicular volume of mice in each experimental group did not change significantly, while the weight of testes and epididymis and testicular and epididymal coefficients of mice in the high-dose group decreased significantly(P<0. 05). The testis seminiferous tubules of every treatment group showed different degrees of degeneration, spermatogenic cell damage, and decreased number of luminal sperm. Meanwhile, medium and high dose treatment groups showed hyperplasia of testicular stromal cells. However, there was no significant pathologic change in penile tissue in every treatment group. The sperm density of mice in the middle and high dose groups was lower than that of the control group(P<0. 05), and the sperm deformity rate in the two groups was significantly higher than that of the control group(P<0. 01), while the sperm motility of every dose treatment group was lower than that of the control(P<0. 05). The testosterone in the testis of the middle and high dose groups were high than the control(P<0. 001), while there was no statistically significant difference of the testosterone among all the groups in the penis(P>0. 05). In addition, the expression of pro-apoptotic genes Caspase-3, Caspase-9, Caspase-12 and Bax in testis tissues of the high-dose group were significantly increased(P<0. 05). CONCLUSION: Procymidone can cause damage to the structure and function of testes, reduce sperm quality, and increase the expression of certain pro-apoptotic genes in adolescent male mice.

[Role of Hippo signaling pathway in mice hypospadias induced by vinclozolin].

OBJECTIVE: To observe the role of Hippo signaling pathway in mice hypospadias induced by vinclozolin. METHODS: After 8 weeks of ICR mice were conceived, they were divided randomly into the treatment and the control groups(10 for each group). Vinclozolin was orally administraed to the pregnant mice in the treatment group with 400 mg/(kg·d) during gestation days 12-18, while those of the control group were treated with equal volume of soybean oil. About 60 days after birth, 20 penises of the mouse offspring were taken randomly from both of the two groups separately. And furthermore, the relative expression abundance of Hippo signaling pathway key genes(Mst1, Lats1, Taz, Yap), androgen receptor gene Ar of all the samples were measured by the real-time quantity reverse transcript polymerase chain reaction(qPCR). The Yes-related protein(Yap) and its phosphorylation in the downstream of Hippo signaling pathway were measured by Western blot. RESULTS: Compared with the male mice of the control group, the anal genital distances of the treatment group were significantly shortened((9. 2501±2. 5504) vs. (16. 1253±1. 3562) mm, P<0. 05), the quality of the penises was significantly less than((0. 0293±0. 0075) vs. (0. 4731±0. 004) g, P<0. 05), the length of the penises was shorter((5. 3875±0. 4524) vs. (12. 4688±1. 2290) mm, P<0. 05), and the diameters of the penises were also less than the control((1. 5513±0. 1158) vs. (2. 6013±0. 1469) mm, P<0. 05). All the genes in the penises of the control group were expressed. The Yap and Ar in the treatment group's penises were not expressed, the expression abundance of Mst1 and Lats1 was higher than that of the control(6. 6097±1. 3188 vs. 0. 3517±0. 1524, 5. 7071±2. 7210 vs. 0. 1235±0. 0658, P<0. 05), and the expression abundance of Taz was lower than that of the control(0. 3937±0. 1519 vs. 3. 2329±0. 4339, P<0. 05). The expression of Yap was decreased in the treatment group(0. 3348±0. 0639 vs. 0. 8023±0. 4275, P<0. 05), and the phosphorylation level of Yap was higher than that of the control group(3. 9940±0. 2177 vs. 0. 3128±0. 0867, P<0. 05). CONCLUSION: In the model of mice hypospadias induced by vinclozolin, the Ar was not expressed, the Hippo signaling pathway was activated and the phosphorylation of Yap was increased. This pathway may play an important role in the model.

mPEG-icariin nanoparticles for treating myocardial ischaemia.

Icariin (ICA), a major active ingredient from Chinese medicine, has unique pharmacological effects on ischaemic heart disease. However, its hydrophobic property limits its administration and leads to poor efficacy. This work aimed to change its hydrophobic property and improve the treatment efficacy. We designed a new nano-drug to increase the ICA delivery. ICA was modified with hydrophilic polyethylene glycol monomethyl ether (mPEG) by a succinic anhydride linker to form a polyethylene glycol-icariin (mPEG-ICA) polymer. The structure of this polymer was identified by Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. The content of ICA in the polymer was 32% as detected by ultraviolet spectrophotometry. mPEG-ICA nanoparticles, of 143.3 nm, were prepared by the dialysis method, and zeta potential was 0.439 mV by dynamic light scattering. The nanoparticles had a spherical shape on transmission electron microscopy. In media with pH 7.4 and 6.8, ICA release from mPEG-ICA nanoparticles after 72 h was about 0.78% and 64.05%, respectively, so the ICA release depended on the release media pH. On MTT and lactate dehydrogenase activity assay, mPEG-ICA nanoparticles could reduce cell damage induced by oxgen-glucose deprivation. Hoechst 33258 staining and TUNEL and AnnexinV-FITC/PI double staining showed that ICA nanoparticles could increase the activity of H9c2 cardiomyocytes under oxgen-glucose deprivation conditions by decreasing apoptosis. ICA modified by hydrophilic mPEG could improve its efficacy.

Gene expression of Hsps in normal and abnormal embryonic development of mouse hindlimbs.

Heat shock proteins (Hsps), which have important biological functions, are a class of highly conserved genetic molecules with the capacity of protecting and promoting cells to repair themselves from damage caused by various stimuli. Our previous studies found that Hsp25, HspB2, HspB3, HspB7, Hsp20, HspB9, HspB10, and Hsp40 may be related to all-trans retinoic acid (atRA)-induced phocomelic and other abnormalities, while HspA12B, HspA14, Trap1, and Hsp105 may be forelimb development-related genes; Grp78 may play an important role in forelimb development. In this study, the embryonic phocomelic, oligodactylic model of both forelimbs and hindlimbs was developed by atRA administered per os to the pregnant mice on gestational day 11, and the expression of 36 members of Hsps family in normal and abnormal development of embryonic hindlimbs was measured by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). It is found that HspA1L, Hsp22, Hsp10, Hsp60, Hsp47, HspB2, HspB10, HspA12A, Apg1, HspB4, Grp78, and HspB9 probably performs a major function in limb development, and HspA13, Grp94 and Hsp110 may be hindlimb development-related genes.