Sanghuangporus vaninii ethanol extract alleviates hyperuricemic renal injury by regulating the uric acid transporters and inhibiting HK-2 apoptosis.

AIM OF THE STUDY: To investigate the acute toxicity of Sanghuangporus ethanol extract (SHEE) on ICR mice and the underlying mechanism of anti-hyperuricemic renal injury. MATERIALS AND METHODS: ICR mice were given a single gavage of 1250, 2500, and 5000 mg/kg SHEE, and the general behavior, mortality, body weight, dietary, and water intake were evaluated within 14 days to determine the acute toxicity level. The hyperuricemic kidney injury model in ICR mice was induced with potassium oxonate (PO) and adenine, and the mice were subsequently treated with SHEE (125, 250, 500 mg/kg). HE and hexamine silver staining (PASM) were used to observe the pathology of the kidney. Biochemical markers were tested by uric acid (UA), creatinine (Cr), blood urea nitrogen (BUN), xanthine oxidase (XOD), alanine transferase (ALT), and aspartate transaminase (AST) kits. An MTT assay was used to measure the effects of SHEE on the proliferation of HK-2 damaged by UA. Western blotting and RT-PCR were used to determine the expression of Bcl-2 family-related proteins and major UA transporters, including URAT1, GLUT9, OAT1, OAT3, and ABCG2, respectively. RESULTS: Firstly, the acute toxicity study data showed that the median lethal dose (LD50) of SHEE was above 5000 mg/kg, and its oral administration was nontoxic at 2500 mg/kg and below. In addition, SHEE alleviated HUA and its renal injury in ICR mice. SHEE reduced the contents of UA, Cr, BUN and XOD in blood and the contents of ALT and AST in the liver. Furthermore, SHEE inhibited the expression of URAT1 and GLUT9 and promoted the expression of OAT1, OAT3, and ABCG2. More importantly, SHEE could downregulate the apoptosis level and caspase-3 activity. CONCLUSIONS: Overall, an oral dose of SHEE below 2500 mg/kg is safe. SHEE inhibits HUA-induced kidney injury by regulating the UA transporters URAT1, GLUT9, OAT1, OAT3 and ABCG2 and inhibiting HK-2 apoptosis.

The Effect of Chinese Agarwood Essential Oil with Cyclodextrin Inclusion against PCPA-Induced Insomnia Rats.

OBJECTIVE: To study the extraction process of agarwood active ingredients (AA) and investigate the safety and effectiveness of AA in the treatment of insomnia rats by nasal administration. METHOD: A ß-cyclodextrin (ß-CD) inclusion compound (a-ß-CD) was prepared from agarwood essential oil (AEO), and the preparation process was optimized and characterized. The safety of AA in nasal mucosa was evaluated through Bufo gargarizans maxillary mucosa and rat nasal mucosa models. Insomnia animal models were replicated by injecting p-chlorophenylalanine (PCPA), conducting behavioral tests, and detecting the expression levels of monoamine neurotransmitters (NE and 5-HT) and amino acids (GABA/Glu) in the rat hypothalamus. RESULTS: The optimum inclusion process conditions of ß-CD were as follows: the feeding ratio was 0.35:1.40 (g:g), the inclusion temperature was 45 °C, the inclusion time was 2 h, and the ICY% and IEO% were 53.78 ± 2.33% and 62.51 ± 3.21%, respectively. The inclusion ratio, temperature, and time are the three factors that have significant effects on the ICY% and IEO% of a-ß-CD. AA presented little damage to the nasal mucosa. AA increased the sleep rate, shortened the sleep latency, and prolonged the sleep time of the rats. The behavioral test results showed that AA could ameliorate depression in insomnia rats to a certain extent. The effect on the expression of monoamine neurotransmitters and amino acids in the hypothalamus of rats showed that AA could significantly reduce NE levels and increase the 5-HT level and GABA/Glu ratio in the hypothalamus of insomnia rats. CONCLUSION: The preparation of a-ß-CD from AEO can reduce its irritation, improve its stability, increase its curative effect, and facilitate its storage and transport. AA have certain therapeutic effects on insomnia. The mechanism of their effect on rat sleep may involve regulating the expression levels of monoamine neurotransmitters and amino acids in the hypothalamus.

Pien-Tze-Huang alleviates CCl4-induced liver fibrosis through the inhibition of HSC autophagy and the TGF-ß1/Smad2 pathway.

Ethnopharmacological relevance: Pien-Tze-Huang (PZH)-a traditional Chinese medicine (TCM) compound-has been employed to treat various liver inflammation and tumors for over 10 decades. Interestingly, most of the pharmacological effects had been validated and explored toward liver ailment along with pro-inflammatory conditions and cancer at the cellular and molecular level to date. Aim of the study: The present study aimed to investigate the therapeutic effect of PZH on autophagy and TGF-ß1 signaling pathways in rats with liver fibrosis and hepatic stellate cell line (HSC). Materials and methods: Male SD rats with carbon tetrachloride (CCl4)-induced liver fibrosis were used as the animal model. Next, PZH treatment was given for 8 weeks. Afterward, the therapeutic effects of PZH were analyzed through a hepatic tissue structure by hematoxylin-eosin (H&E), Van Gieson (VG) staining, and transmission electron microscopy (TEM), activity of ALT and AST by enzyme-associated immunosorbent assay as well. Subsequently, mRNA and protein expression were examined by quantitative polymerase chain reaction (qPCR), Western blotting, and immunohistochemistry (IHC). Then, the cell vitality of PZH-treated HSC and the expression of key molecules prevailing to autophagy were studied in vitro. Meanwhile, SM16 (a novel small molecular inhibitor which inhibits TGFß-induced Smad2 phosphorylation) was employed to confirm PZH's effects on the proliferation and autophagy of HSC. Results: PZH pharmacologically exerted anti-hepatic fibrosis effects as demonstrated by protecting hepatocytes and improving hepatic function. The results revealed the reduced production of extracellular collagen by adjusting the balance of matrix metalloproteinase (MMP) 2, MMP9, and tissue inhibitor of matrix metalloproteinase 1 (TIMP1) in PZH-treated CCl4-induced liver fibrosis. Interestingly, PZH inhibited the activation of HSC by down-regulating TGF-ß1 and phosphorylating Smad2. Furthermore, PZH down-regulated yeast Atg6 (Beclin-1) and microtubule-associated protein light chain 3 (LC3) toward suppressing HSC autophagy, and PZH exhibited similar effects to that of SM16. Conclusion: To conclude, PZH alleviated CCl4-induced liver fibrosis to reduce the production of extracellular collagen and inhibiting the activation of HSC. In addition, their pharmacological mechanisms related to autophagy and TGF-ß1/Smad2 signaling pathways were revealed for the first time.

Anti-Müllerian Hormone Inhibits FSH-Induced Cumulus Oocyte Complex In Vitro Maturation and Cumulus Expansion in Mice.

Anti-Müllerian hormone (AMH) is secreted by the ovaries of female animals and exerts its biological effects through the type II receptor (AMHR2). AMH regulates follicular growth by inhibiting the recruitment of primordial follicles and reducing the sensitivity of antral follicles to FSH. Despite the considerable research on the actions of AMH in granulosa cells, the effect of AMH on the in vitro maturation of oocytes remains largely unknown. In the current study, we showed that AMH is only expressed in cumulus cells, while AMHR2 is produced in both cumulus cells and oocytes. AMH had no significant effect on COCs nuclear maturation, whereas it inhibited the stimulatory effects of FSH on COCs maturation and cumulus expansion. Moreover, AMH treatment effectively inhibited the positive effect of FSH on the mRNA expressions of Hyaluronan synthase 2 (Has2), Pentraxin 3 (Ptx3), and TNF-alpha-induced protein 6 (Tnfaip 6) genes in COCs. In addition, AMH significantly decreased the FSH-stimulated progesterone production, but did not change estradiol levels. Taken together, our results suggest that AMH may inhibit the effects of FSH-induced COCs in vitro maturation and cumulus expansion. These findings increase our knowledge of the functional role of AMH in regulating folliculogenesis.

Novel functional mutation of the PDIA3 gene affects milk composition traits in Chinese Holstein cattle.

Protein disulfide isomerase family A member 3 (PDIA3) is a multifunctional protein, and it plays a vital role in modulating various cell biological functions under physiological and pathological conditions. Our previous study on Mediterranean buffalo demonstrated that PDIA3 is a potential candidate gene associated with milk yield based on genome-wide association study analysis. However, the genetic effects of the PDIA3 gene on milk performance in dairy cattle and the corresponding mechanism have not been documented. This study aims to explore the genetic effects of PDIA3 polymorphisms on milk production traits in 362 Chinese Holstein cattle. The results showed that 4 SNPs were identified from the 5' untranslated region of the PDIA3 gene in the studied population, of which 2 SNPs (g.-1713 C>T and g.-934 G>A) were confirmed to be significantly associated with milk protein percentage, whereas g.-434 C>T was significantly associated with milk fat percentage. Notably, linkage disequilibrium analysis indicated that 3 SNPs (g.-1713 C>T, g.-934 G>A, and g.-695 A>C) formed one haplotype block, which was found to be significantly associated with milk protein percentage. The luciferase assay demonstrated that allele C of g.-434 C>T exhibited a higher promotor activity compared with allele T, suggesting that g.-434 C>T might be a potential functional mutation affecting PDIA3 expression. Furthermore, overexpression of the PDIA3 gene was found to induce higher levels of triglyceride and BODIPY fluorescence intensity. In addition, PDIA3 overexpression was also found to positively regulate the synthesis and secretion of α-casein, ß-casein, and κ-casein, whereas knockdown of this gene showed the opposite effects. In summary, our findings revealed significant genetic effects of PDIA3 on milk composition traits, and the identified SNP and the haplotype block might be used as genetic markers for dairy cow selected breeding.

Synergistic Regulatory Effect of Inhibin and Anti-Müllerian Hormone on Fertility of Mice.

Inhibin (INH) and anti-Müllerian hormone (AMH) are essential in ovarian folliculogenesis and play an inhibitory role in mammalian fertility. However, the interactive effect of INH and AMH on the animal reproduction remains unknown. This study aimed to determine the possible interaction and synergy between INH and AMH in steroidogenesis by primary granulosa cells, and investigate their synergistic effect on fertility in mice. In in vitro granulosa cell culture system, we found that the treatment of either INHA or AMH had no significant effect on basal estradiol and progesterone production, whereas both significantly attenuated FSH-induced steroid hormone secretion. Importantly, combined treatment with INHA and AMH showed additive inhibitory effect on FSH-induced estradiol and progesterone production, accompanying a significant downregulation in the expression of FSH-stimulated CYP19A1, HSD3B, CYP11A1, StAR transcripts. The interrelationship of INH and AMH combinations was further investigated through active immune neutralization strategy. Female mice were immunized against INH and AMH eukaryotic expression plasmids, and the litter size was recorded after successfully mating. We observed that both INH and AMH plasmids were able to induce either anti-AMH or anti-INH antibodies in the immunized mice. In comparison with the control group, co-immunization with INH and AMH plasmids induced higher levels of estradiol, resulting in more litter size. Moreover, there was no significant difference on the offspring's weight between each group. Collectively, the results of the present study suggest that INH and AMH have synergistic effect in regulating steroidogenesis and the litter size in mice.

Robust three-dimensional nanotube-in-micropillar array electrodes to facilitate size independent electroporation in blood cell therapy.

Blood is an attractive carrier for plasmid and RNA-based medicine in cell therapy. Electroporation serves as a favorable delivery tool for simple operation, quick internalization, minimum cell culture involvement, and low contamination risk. However, the delivery outcome of electroporation heavily depends on the treated cells such as their type, size, and orientation to the electric field, not ideal for highly heterogeneous blood samples. Herein, a new electroporation system was developed towards effective transfection to cells in blood regardless of their large diversity. By coupling replica molding and infiltration-coating processes, we successfully configured a three-dimensional electrode comprised of a polymer micropillar array on which carbon nanotubes (CNTs) are partially embedded. During electroporation, cells sag between micropillars and deform to form a conformal contact with their top and side surfaces. The implanted CNTs not only provide a robust conductive coating for polymer micropattern but also have their protruded ends face the cell membrane vertically everywhere with maximum transmembrane potential. Regardless of their largely varied sizes and random dispersion, both individual blood cell type and whole blood samples were effectively transfected with plasmid DNA (85% after 24 h and 95% after 72 h, or 2.5-3.0 folds enhancement). High-dose RNA probes were also introduced, which regulate better the expression levels of exogenous and endogenous genes in blood cells. Besides its promising performance on non-viral delivery routes to cell-related studies and therapy, the involved new fabrication method also provides a convenient and effective way to construct flexible electronics with stable micro/nano features on the surface.

Microfluidic Electroporation Coupling Pulses of Nanoseconds and Milliseconds to Facilitate Rapid Uptake and Enhanced Expression of DNA in Cell Therapy.

Standard electroporation with pulses in milliseconds has been used as an effective tool to deliver drugs or genetic probes into cells, while irreversible electroporation with nanosecond pulses is explored to alter intracellular activities for pulse-induced apoptosis. A combination treatment, long nanosecond pulses followed by standard millisecond pulses, is adopted in this work to help facilitate DNA plasmids to cross both cell plasma membrane and nuclear membrane quickly to promote the transgene expression level and kinetics in both adherent and suspension cells. Nanosecond pulses with 400-800 ns duration are found effective on disrupting nuclear membrane to advance nuclear delivery of plasmid DNA. The additional microfluidic operation further helps suppress the negative impacts such as Joule heating and gas bubble evolution from common nanosecond pulse treatment that lead to high toxicity and/or ineffective transfection. Having appropriate order and little delay between the two types of treatment with different pulse duration is critical to guarantee the effectiveness: 2 folds or higher transfection efficiency enhancement and rapid transgene expression kinetics of GFP plasmids at no compromise of cell viability. The implementation of this new electroporation approach may benefit many biology studies and clinical practice that needs efficient delivery of exogenous probes.

Diethylstilbestrol exposure disrupts mouse oocyte meiotic maturation in vitro through affecting spindle assembly and chromosome alignment.

An adverse tendency induced by the environmental estrogens in female reproductive health is one serious problem worldwide. Diethylstilbestrol (DES), as a synthetic estrogen, is still used as an animal growth stimulant in terrestrial livestock and aquaculture illegally. It has been reported to negatively affect ovarian function and oogenesis. Nevertheless, the mechanism and toxicity of DES on oocyte meiotic maturation are largely unknown. Herein, we found that DES (40 µM) intervened in mouse oocyte maturation and first polar body extrusion (PBE) was decreased in vitro. Cell cycle analysis showed meiotic process was disturbed with oocytes arrested at metaphase I (MI) stage after DES exposure. Further study showed that DES exposure disrupted the spindle assembly and chromosome alignment, which then continuously provoke the spindle assemble checkpoint (SAC). We also observed that the acetylation levels of α-tubulin were dramatically increased in DES-treated oocytes. In addition, the dynamics of actin were also affected. Moreover, the distribution patterns of estrogen receptor α (ERα) were altered in DES-treated oocyte, as indicated by the significant signals accumulation in the spindle area. However, ERα inhibitor failed to rescue the defects of oocyte maturation caused by DES. Of note, the same phenomenon was observed in estrogen-treated oocytes. Collectively, we showed that DES exposure lead to the oocyte meiotic failure via impairing the spindle assembly and chromosome alignment. Our research is helpful to understand how environmental estrogen affects female germ cells and contribute to design the potential therapies to preserve fertility especially for occupational exposure.

Doxorubicin Exposure Affects Oocyte Meiotic Maturation through DNA Damage-Induced Meiotic Arrest.

Developments in chemotherapeutics have enhanced the survival rate of cancer patients, however, adverse effects of chemotherapeutics on ovarian functions causes the fertility loss in young female cancer patients. Doxorubicin (DOX), as an anthracycline antitumor antibiotic, is extensively used to cure various malignancies. Recent studies have suggested that DOX can cause ovarian damage and affect the oocyte maturation, nevertheless the mechanism by which DOX on oocytes meiosis is poorly understood. In this study, we explored the mechanism for DOX-induced oocytes meiotic failure in vitro at human relevant exposure levels and time periods. Results described that DOX (100 nM) can interrupt the mouse oocytes meiotic maturation directly with reduced first polar body extrusion. Cell cycle analysis showed that most oocytes were arrested at metaphase I (MI) stage. However, DOX treatment had no effect on spindle structure but chromosomal misalignment. We observed that kinetochore-microtubule structure was affected and the spindle assemble checkpoint was provoked after DOX treatment. Moreover, severe DNA damage was found in DOX-treated oocytes indicated by the positive γ-H2A.X foci signal, which then may trigger oocytes early apoptosis. Besides, metaphase II oocytes with disorganized spindle morphologies and misaligned chromosomes were observed after DOX treatment. In conclusion, DOX have the potential to disrupt oocyte meiotic maturation through DNA damage induced meiotic arrest mediated by spindle assemble checkpoint activation. These findings can contribute to design the new therapies to alleviate DNA damage to preserve fertility for young female cancer patients with chemotherapeutics.

Pien-Tze-Huang ameliorates hepatic fibrosis via suppressing NF-κB pathway and promoting HSC apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Pien Tze Huang (PZH), a Chinese herbal formula, has various forms of pharmacological activity including anti-inflammation, liver protection and anti-tumor. AIM OF THE STUDY: To investigate the anti-hepatic fibrosis effect of PZH and its potential mechanisms on experimental animal model of hepatic fibrosis and hepatic stellate cell (HSC). MATERIAL AND METHODS: Rats were intraperitoneally administered with CCl4 to induce hepatic fibrosis and were simultaneously treated with PZH. Liver pathology were observed by hematoxylin and eosin (H&E) staining and Masson staining. Serum pro-inflammatory cytokines were determined by enzyme-linked immunosorbent assay (ELISA) kits. Moreover, the effects of PZH on HSC proliferation and apoptosis were assessed via MTT, flow cytometry and immunofluorescence analysis. Nuclear factor-κB (NF-κB) pathway activation and Bcl-2 family members were evaluated by reverse transcription quantitative polymerase chain reaction (real-time PCR) and western blotting. RESULT: PZH significantly alleviated CCl4-induced liver injury, inflammation and fibrogenesis in rats. PZH also markedly decreased the production of hepatic-fibrosis biomarker, including ALT, AST, IV collagen and PCIII (Procollagen III), as well as inflammatory cytokines such as IL-1ß, IL-6 and TNF-α. Importantly, PZH strongly inhibited HSC proliferation correlated with cell apoptosis induction as evidenced by modulating Bcl-2 family members and caspase activity. Moreover, PZH administration significantly increased the expression IκB-α, an inhibitor of NF-κB and suppressed expression of anti-apoptotic genes (Bcl-2, etc.). Collectively, these results suggested that PZH could promote HSC apoptosis through inhibiting NF-κB signaling pathway. CONCLUSION: Our study demonstrates that PZH ameliorates hepatic fibrosis and inflammation, chiefly through suppressing the NF-κB pathway and promoting HSC apoptosis. PZH is more likely to be a promising antifibrotic agent in chronic disease.

A new polyoxygenated abietane diterpenoid from the rattans of Bauhinia championii (Benth.) Benth.

A new polyoxygenated abietane diterpenoid, bauchampine A (1), together with seven known compounds (2-8), were isolated from the rattans of Bauhinia championii (Benth.) Benth. The structure of 1 was elucidated by extensive spectroscopic methods and the known compounds were identified by comparison with the data reported in the literature. New compound 1 was evaluated for its anti-rheumatoid arthritis activity via examining its anti-proliferative effect on synoviocytes in vitro. Compound 1 exhibited inhibitory effect on the proliferation of synoviocytes with IC50 value comparable to that of methotrexate.