[Mechanism of Liangfang Wenjing Decoction in treatment of hypoxia on endometriosis with cold coagulation and blood stasis by regulating CHCHD4 expression].

To explore the mechanism of Liangfang Wenjing Decoction regulating coiled-coil-helix coiled-coil-helix domain containing 4(CHCHD4) in the treatment of hypoxia on endometriosis(EMs) with cold coagulation and blood stasis. The rat model of cold coagulation and blood stasis syndrome was prepared by the ice-water bath method, and then the EMs model was established by autologous intimal transplantation. The rats were randomly divided into model group, low, medium, and high(4.7, 9.4, and 18.8 g·kg~(-1)) dose groups of Liangfang Wenjing Decoction, Shaofu Zhuyu Decoction group, and sham group, with 10 rats in each group. The rats were given intragastric administration for four weeks. During the modeling, the general condition and vaginal smear of rats were observed, and the blood flow of ears and uterus were detected by laser speckle contrast imaging(LSCI) to judge the syndrome of cold coagulation and blood stasis. After the administration, the general condition of the rats was observed, and the area of ectopic lesions was measured by caliper. The localization and expression of CHCHD4 and hypoxia inducible factors-1α(HIF-1α) were detected by immunohistochemistry, and the mRNA and protein expressions of CHCHD4 and HIF-1α were detected by real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot. The primary culture of ectopic endometrial stromal cells(ESCs) from EMs patients was performed, and the CHCHD4 overexpression plasmid was constructed and transfected to establish the ESCs model of CHCHD4 overexpression. The cells were divided into the control group, CHCHD4 overexpression group, CHCHD4 overexpression+control serum group, and CHCHD4 overexpression+Liangfang Wenjing Decoction serum group. The protein expression of CHCHD4 and HIF-1α was detected by Western blot, and the glucose consumption and lactic acid level were detected. The cell proliferation was detected by MTT assay. The experiment found that compared with normal rats, the modeling rats showed symptoms of cold coagulation and blood stasis, such as mental malaise, reduced diet and drinking water, disordered estrous cycle, and blocked blood circulation in ears and uterine microvessels. Compared with the sham group, the ectopic lesions in the model group were uplifted, and the mRNA and protein expressions of CHCHD4 and HIF-1α were significantly increased(P<0.05). Compared with the model group, the symptoms of cold coagulation and blood stasis in each treatment group were improved, and the area of ectopic lesions was significantly reduced(P<0.05 or P<0.01). The mRNA and protein expression levels of CHCHD4 and HIF-1α were significantly decreased(P<0.05 or P<0.01). In the cell model, compared with the control group, the expression of CHCHD4, HIF-1α protein, glucose consumption, lactic acid level, and cell proliferation activity in the CHCHD4 overexpression group were significantly increased(P<0.01). Compared with the CHCHD4 overexpression group, there was no significant change in each index in the control serum group, while the protein expression of CHCHD4 and HIF-1α in the Liangfang Wenjing Decoction serum group was decreased significantly(P<0.05 or P<0.01). The glucose consumption, lactic acid level, and cell proliferation activity decreased significantly(P<0.01). It can be seen from the above that the therapeutic effect of Liangfang Wenjing Decoction on EMs with cold coagulation and blood stasis might be related to reducing the expression of CHCHD4 and then improving the hypoxia of ectopic lesions and ectopic ESCs.

[Effect of Liangfang Wenjing Decoction on expression of key glycolytic enzymes in uterus and ovaries of rats with coagulating cold and blood stasis syndrome].

This study aimed to investigate the relationship between coagulating cold and blood stasis syndrome and glycolysis, and observe the intervention effect of Liangfang Wenjing Decoction(LFWJD) on the expression of key glycolytic enzymes in the uterus and ovaries of rats with coagulating cold and blood stasis. The rat model of coagulating cold and blood stasis syndrome was established by ice-water bath. After modeling, the quantitative scoring of symptoms were performed, and according to the scoring results, the rats were randomly divided into a model group and LFWJD low-, medium-and high-dose groups(4.7, 9.4, 18.8 g·kg~(-1)·d~(-1)), with 10 in each group. Another 10 rats were selected as the blank group. After 4 weeks of continuous administration by gavage, the quantitative scoring of symptoms was repeated. Laser speckle flowgraphy was used to detect the changes of microcirculation in the ears and uterus of rats in each group. Hematoxylin-eosin(HE) staining was used to observe the pathological morphology of uterus and ovaries of rats in each group. The mRNA and protein expressions of pyruvate dehydrogenase kinase 1(PDK1), hexokinase 2(HK2) and lactate dehydrogenase A(LDHA) in the uterus and ovaries of rats were examined by real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot, respectively. The rats in the model group showed signs of coagulating cold and blood stasis syndrome, such as curl-up, less movement, thickened veins under the tongue, and reduced blood perfusion in the microcirculation of the ears and uterus, and HE staining revealed a thinning of the endometrium with disorganized arrangement of epithelial cells and a decrease in the number of ovarian follicles. Compared with the model group, the treatment groups had alleviated coagulating cold and blood stasis, which was manifested as red tongue, reduced nail swelling, no blood stasis at the tail end as well as increased blood perfusion of the microcirculation in the ears and uterus(P<0.05 or P<0.01). Among the groups, the LFWJD medium-and high-dose groups had the most significant improvement in coagulating cold and blood stasis, with neatly arranged columnar epithelial cells in uterus, and the number of ovarian follicles was higher than that in the model group, especially mature follicles. The mRNA and protein expressions of PDK1, HK2, LDHA in uterus and ovaries were up-regulated in the model group(P<0.05 or P<0.01), while down-regulated in LFWJD medium-and high-dose groups(P<0.05 or P<0.01). The LFWJD low-dose group presented a decrease in the mRNA expressions of PDK1, HK2 and LDHA in uterus and ovaries as well as in the protein expressions of HK2 and LDHA in uterus and HK2 and PDK1 in ovaries(P<0.05 or P<0.01). The therapeutic mechanism of LFWJD against coagulating cold and blood stasis syndrome is related to the down-regulation of key glycolytic enzymes PDK1, HK2 and LDHA, and the inhibition of glycolytic activities in uterus and ovaries.

CHCHD2 Regulates Mitochondrial Function and Apoptosis of Ectopic Endometrial Stromal Cells in the Pathogenesis of Endometriosis.

Endometriosis is a disease that involves dysfunction of mitochondria, imbalance of proliferation, and apoptosis. Coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) is a major mitochondrial protein which could regulate the mitochondrial function and apoptosis in various tumor cells, promote migration and then lead to tumor progression. This study aimed to explore the role of CHCHD2 on endometriosis. We investigated the expression of CHCHD2 in ectopic and eutopic endometrium tissues of patients with endometriosis and normal endometrium tissues. Furthermore, CHCHD2 was downregulated to explore the corresponding change of mitochondrial function and morphology, mitochondrial-mediated apoptosis pathway, and proliferation and migration of ectopic endometrial stromal cells. Our results demonstrated that the mRNA and protein expression levels of CHCHD2 were significantly increased in eutopic and ectopic endometrium tissues compared with the normal endometrium tissues. The knockdown of CHCHD2 could cause mitochondrial dysfunction, including the opening of mitochondrial permeability transition pore, loss of mitochondrial membrane potential and the release of cytochrome c, and morphological damage. In addition, CHCHD2 down-expression could also lead to inhibition of cell proliferation, decrease of migration ability, and aggravation of mitochondrial-mediated apoptosis. Together, these findings suggest that increased expression of CHCHD2 in endometriotic tissues may contribute to the pathogenesis of endometriosis via regulating mitochondrial function and apoptosis, and CHCHD2 may be a potential target for interrupting the development of endometriosis.

Effects of modified Wenjing decoction on microcirculation in reproductive organs in rats with symptom patterns of cold coagulation and blood stasis.

OBJECTIVE: To investigate the relationship between symptom patterns of cold coagulation and blood stasis (CCBS) and microcirculation disturbance. In addition, we determined the efficacy of modified Wenjing decoction (WJD) for the treatment of CCBS. METHODS: CCBS was induced in rats with an ice-water bath treatment. The ovarian function, microvascular and circulatory status of reproductive organs, and function of local microvascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) were evaluated. RESULTS: Ovarian dysfunction was observed in the rats with CCBS. It was characterized by the presence of an estrous cycle disorder and a decrease in reproductive hormone levels. Microvascular circulation disorders were associated with an imbalance in vasoconstriction, relaxation substances, nitric oxide, abnormal blood flow in whole blood, and decreased blood flow in the auricle and uterus. VECs were damaged, and VSMCs contracted and proliferated in ovarian and uterine tissues. CONCLUSION: Our findings suggest that the dysfunctional reproductive organs observed in gynecological CCBS may be closely related to the microcirculation disturbance of local tissues, microvascular contraction, and vascular remodeling. Modified WJD can be used to treat CCBS by improving microcirculation in reproductive organs.

Effect of cold stress on ovarian & uterine microcirculation in rats and the role of endothelin system.

BACKGROUND: Cold, an environmental factor, induces many reproductive diseases. It is known that endothelin (ET) is a potent vasoconstrictor, and cold stress can increase the expression of ET and its receptors. The cold stress rat model was developed to examine two parameters: (1) the effects of cold stress on ovarian and uterine morphology, function, and microvascular circulation and (2) possible mechanisms of ET and its receptors involved in cold stress-induced menstruation disorders. METHODS: The rat cold stress model was prepared with an ice water bath. The estrous cycle was observed by methylene blue and hematoxylin and eosin (H&E) staining. Serum estradiol 2 (E2), testosterone (T), progesterone (P) were detected by radioimmunoassay. Hemorheology indices were measured. The real-time blood flow of auricle and uterine surfaces was measured. Expressions of CD34 and α-SMA in ovarian and uterine tissues were detected by immunohistochemistry. ET-1 contents in serum were tested, and expressions of ET-receptor types A and B (ET-AR and ET-BR) in ovarian tissues were detected via Western blotting. RESULTS: Cold stress extended the estrous cycle, thereby causing reproductive hormone disorder, imbalance of local endothelin/nitric oxide expression, and microcirculation disturbance. Cold-stress led to up-regulation of ET-AR expression and protein and down-regulation of ET-BR expression in rats. CONCLUSIONS: This study suggests that the reason for cold stress-induced dysfunction in reproductive organs may be closely related to the imbalance of ET-1 and its receptor expressions, leading to microvascular circulation disorders in local tissues.

Pharmacokinetics, distribution and anti-tumor efficacy of liposomal mitoxantrone modified with a luteinizing hormone-releasing hormone receptor-specific peptide.

BACKGROUND: A previous study developed a novel luteinizing hormone-releasing hormone (LHRH) receptor-targeted liposome. The aim of this study was to further assess the pharmacokinetics, biodistribution, and anti-tumor efficacy of LHRH receptor-targeted liposomes loaded with the anticancer drug mitoxantrone (MTO). METHODS: Plasma and tissue distribution profiles of LHRH receptor-targeted MTO-loaded liposomes (LHRH-MTO-LIPs) were quantified in healthy mice or a xenograft tumor nude mouse model of MCF-7 breast cancer, and were compared with non-targeted liposomes and a free-drug solution. RESULTS: The LHRH-MTO-LIPs demonstrated a superior pharmacokinetic profile relative to free MTO. The first target site of accumulation is the kidney, followed by the liver, and then the tumor; maximal tumor accumulation occurs at 4 h post-administration. Moreover, the LHRH-MTO-LIPs exhibited enhanced inhibition of MCF-7 breast cancer cell growth in vivo compared with non-targeted MTO-loaded liposomes (MTO-LIPs) and free MTO. CONCLUSION: The novel LHRH receptor-targeted liposome may become a viable platform for the future targeted treatment of cancer.

Synergistic cytotoxicity and mechanism of caffeine and lysozyme on hepatoma cell line HepG2.

The influences of caffeine, lysozyme and the joint application of them on the hepatoma cell line HepG2 proliferation inhibition and cell apoptosis were observed by 3-(4, 5-dimethyl-2-thiazyl)-2, 5-diphenyl-2H-tetrazolium bromide assay and Hoechst 33342, which showed the proliferation inhibition rate of the joint application on HepG2 cells was 47.21%, significantly higher than caffeine or lysozyme, and the joint application promoted the apoptosis of HepG2 cells obviously. Van't Hoff classical thermodynamics formula, the Föster theory of non-radiation energy transfer and fluorescence phase diagram were used to manifest that the process of lysozyme binding to caffeine followed a two-state model, which was spontaneous at low temperature driven by enthalpy change, and the predominant intermolecular force was hydrogen bonding or Van der Waals force to stabilize caffeine-lysozyme complex with the distance 5.86nm. The attenuated total reflection-Fourier transform infrared spectra indicated that caffeine decreased the relative contents of α-helix and ß-turn, which inferred the structure of lysozyme tended to be "loose". Synchronous fluorescence spectra and ultraviolet spectra supported the above conclusion. The amino acid residues in the cleft of lysozyme were exposed and electropositivity was increased attributing to the loose structure, which were conducive to increasing caffeine concentration on the HepG2 cell surface by electrostatic interaction to show synergistic effect.

Iron improving bio-char derived from microalgae on removal of tetracycline from aqueous system.

Novel magnetic carbonaceous bio-char was hydrothermal prepared from microalgae under different loadings of iron and its structures and surface chemistry were characterized with Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and nitrogen adsorption-desorption isotherm (BET). The morphology of bio-char changed from sheet to particle as iron loading increased and its surface area also increased. When 3.0 g of dried microalgae and 6.0 mmol iron salt ((NH4)2SO4·FeSO4·6H2O) were mixed and treated, the obtained bio-char possessing the highest amount of oxygen-containing functional groups resulted in the best adsorption performance on tetracycline (TC). This adsorption process was fitted to Langmuir adsorption isotherm and the maximum adsorption capacity was 95.86 mg/g, which is higher than other bio-char reported. The iron loading contributed to the higher adsorption capacity of bio-char, which may be due to three factors, the high surface area, more hydrogen bonding, and bridging effects of the structural Fe for TC. Our data suggest that bio-char may have more important role in stabilization of pollutants in the environment.

Synthesis, chemical reactivity as Michael acceptors, and biological potency of monocyclic cyanoenones, novel and highly potent anti-inflammatory and cytoprotective agents.

Novel monocyclic cyanoenones examined to date display unique features regarding chemical reactivity as Michael acceptors and biological potency. Remarkably, in some biological assays, the simple structure is more potent than pentacyclic triterpenoids (e.g., CDDO and bardoxolone methyl) and tricycles (e.g., TBE-31). Among monocyclic cyanoenones, 1 is a highly reactive Michael acceptor with thiol nucleophiles. Furthermore, an important feature of 1 is that its Michael addition is reversible. For the inhibition of NO production, 1 shows the highest potency. Notably, its potency is about three times higher than CDDO, whose methyl ester (bardoxolone methyl) is presently in phase III clinical trials. For the induction of NQO1, 1 also demonstrated the highest potency. These results suggest that the reactivity of these Michael acceptors is closely related to their biological potency. Interestingly, in LPS-stimulated macrophages, 1 causes apoptosis and inhibits secretion of TNF-α and IL-1ß with potencies that are higher than those of bardoxolone methyl and TBE-31.

Retinoid X receptor and peroxisome proliferator-activated receptor-gamma agonists cooperate to inhibit matrix metalloproteinase gene expression.

INTRODUCTION: We recently described the ability of retinoid X receptor (RXR) ligand LG100268 (LG268) to inhibit interleukin-1-beta (IL-1-beta)-driven matrix metalloproteinase-1 (MMP-1) and MMP-13 gene expression in SW-1353 chondrosarcoma cells. Other investigators have demonstrated similar effects in chondrocytes treated with rosiglitazone, a ligand for peroxisome proliferator-activated receptor-gamma (PPARgamma), for which RXR is an obligate dimerization partner. The goals of this study were to evaluate the inhibition of IL-1-beta-induced expression of MMP-1 and MMP-13 by combinatorial treatment with RXR and PPARgamma ligands and to investigate the molecular mechanisms of this inhibition. METHODS: We used real-time reverse transcription-polymerase chain reaction to measure LG268- and rosiglitazone-mediated inhibition of MMP gene transcription in IL-1-beta-treated SW-1353 chondrosarcoma cells. An in vitro collagen destruction assay was a functional readout of MMP collagenolytic activity. Luciferase reporter assays tested the function of a putative regulatory element in the promoters of MMP-1 and MMP-13, and chromatin immunoprecipitation (ChIP) assays detected PPARgamma and changes in histone acetylation at this site. Post-translational modification of RXR and PPARgamma by small ubiquitin-like modifier (SUMO) was assayed with immunoprecipitation and Western blot. RESULTS: Rosiglitazone inhibited MMP-1 and MMP-13 expression in IL-1-beta-treated SW-1353 cells at the mRNA and heterogeneous nuclear RNA levels and blunted IL-1-beta-induced collagen destruction in vitro. Combining LG268 and rosiglitazone had an additive inhibitory effect on MMP-1 and MMP-13 transcription and collagenolysis. IL-1-beta inhibited luciferase expression in the MMP reporter assay, but rosiglitazone and LG268 had no effect. ChIP indicated that treatment with IL-1-beta, but not LG268 and rosiglitazone, increased PPARgamma at the proximal promoters of both MMPs. Finally, rosiglitazone or LG268 induced 'cross-SUMOylation' of both the target receptor and its binding partner, and IL-1-beta-alone had no effect on SUMOylation of RXR and PPARgamma but antagonized the ligand-induced SUMOylation of both receptors. CONCLUSIONS: The PPARgamma and RXR ligands rosiglitazone and LG268 may act through similar mechanisms, inhibiting MMP-1 and MMP-13 transcription. Combinatorial treatment activates each partner of the RXR:PPARgamma heterodimer and inhibits IL-1-beta-induced expression of MMP-1 and MMP-13 more effectively than either compound alone. We conclude that the efficacy of combined treatment with lower doses of each drug may minimize potential side effects of treatment with these compounds.