Effect of Liangfang Wenjing Decoction on expression of key glycolytic enzymes in uterus and ovaries of rats with coagulating cold and blood stasis syndrome / 中国中药杂志

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.

Regulation of Wenjingtang on HIF-1α Expression and Mitochondrial Function of Ectopic Endometrial Stromal Cells in Patients with Endometriosis / 中国实验方剂学杂志

ObjectiveThis study investigated the mechanism of Wenjingtang in the prevention and treatment of endometriosis （EMT） from the perspective of regulating hypoxia stress and mitochondrial function. MethodPrimary human endometrial stromal cells （ESCs） form ectopic endometrial tissues were isolated and cultured， the cells were divided into control group （Control）， 5% control serum group （5% KBXQ）， 10% control serum group （10% KBXQ）， 5% Wenjingtang serum group （5% WJTXQ） and 10% Wenjingtang serum group （10% WJTXQ）. ESCs in different groups were detected for proliferation by methyl thiazolyl tetrazolium （MTT） assay， mRNA and protein expression of hypoxia inducible factor-1α （HIF-1α） by real-time fluorescent quantitative polymerase chain reaction （Real-time PCR） and Western blot analysis， mitochondrial ultrastructure by transmission electron microscope， mitochondrial function ［mitochondrial membrane potential， mitochondrial membrane potential（MMP） and cytochrome C（Cyt C） content］ and apoptosis （cell membrane permeability， nuclear fluorescence intensity， nuclear size and cell counts） by high content screening （HCS） assay， apoptosis rate by flow cytometry， and proteins of B-cell lymphoma-2 （Bcl-2） associated X （Bax）， Bcl-2 and cleaved cysteinyl aspartate specific proteinase-3 （cleaved Caspase-3） by Western blot. ResultCompared with Control group， the 5% KBXQ and 10% KBXQ groups showed increased cell viability （P<0.01）， there was no significant change in HIF-1α mRNA and protein expression， transmission electron microscopy showed that the mitochondrial cristae were obvious and the inner and outer membranes of mitochondria were clear， HCS multichannel fluorescence staining showed that there were no significant changes in the expression of MMP， Cyt C and cell membrane permeability， and the nuclei showed uniform light staining， there were no significant changes in apoptosis rate， cleaved Caspase-3 protein expression and Bax/Bcl-2 ratio. Compared with Control group and corresponding concentration KBXQ group， the 5% WJTXQ and 10% WJTXQ group showed decreased cell viability （P<0.01） and HIF-1α mRNA and protein levels （P<0.05，P<0.01）， the ultrastructure of mitochondria was destroyed， some mitochondria were swollen and the cristae were blurred， moreover， decreased MMP and up-regulated Cyt C release （P<0.05，P<0.01）， increased cell membrane permeability （P<0.01）， and apoptosis characteristics included nuclear pyknosis， DNA agglutination in nucleus and decrease of cell numbers were observed （P<0.05，P<0.01）， increased apoptosis rate （P<0.01）， which was consistent with the results of HCS analysis， and up-regulated expression levels of cleaved Caspase-3 protein and Bax/Bcl-2 ratio （P<0.05，P<0.01）. ConclusionIn conclusion， the results suggest that Wenjingtang can improve hypoxia stress via down-regulating HIF-1α expression in ectopic ESCs， and inhibit cell proliferation， reduce mitochondrial biological activity and induce apoptosis， which might be the internal mechanism of Wenjingtang in preventing and treating EMT.

Mechanism of Wenjingtang on Improving Endometriosis in Rats Based on Regulating HIF-1α Expression / 中国实验方剂学杂志

Objective:To observe the effect of Wenjingtang on the expression of hypoxia inducible factor-1α （HIF-1α） and ultrastructure of mitochondria in rats with endometriosis （EMs）， and to explore the mechanism of Wenjingtang in the treatment of EMs. Method:The EMs model rat was established by autogenous intimal transplantation， and the volume of ectopic lesions was measured by small animal ultrasound imaging system. According to the volume of ectopic lesions， rats successfully modeled were randomly divided into model group， low， middle ang high dose Wenjingtang group （4.85， 9.7， 19.4 g·kg-1） and gestrinone group （0.25 mg·kg-1）， 10 in each group， another 10 rats were recruited in a sham operation group. After 6 weeks of drug treatment， the volume of ectopic lesions was measured by ultrasound imaging system and caliper， the morphology of ectopic endometrium was observed by hematoxylin-eosin （HE） staining， the levels of interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α） and transforming growth factor-β1 （TGF-β1） in peritoneal fluid were measured by enzyme linked immunosorbent assay （ELISA）， the mRNA and protein expression of HIF-1α in eutopic or ectopic endometrial tissues were detected by Real-time fluorescent quantitative polymerase chain reaction （Real-time PCR） and Western blot， and the ultrastructure of mitochondria in ectopic endometrium was observed by transmission electron microscope （TEM）. Result:Compared with sham operation group， ectopic lesions were found in model group， showing vesicular structure， and the typical endometrial tissue morphology was found in the lesions. The contents of IL-1β， TNF-α， TGF-β1 and the expression of HIF-1α mRNA and protein were significantly increased than those in the sham operation group （P<0.05，P<0.01）. TEM showed that the number of mitochondria in the cytoplasm of ectopic endometrium increased and the structure was intact. Compared with model group， the volume of ectopic focus was significantly decreased in all Wenjingtang groups （P<0.01）， and the results of ultrasonic examination were basically consistent with those of caliper measurement， HE staining showed that the columnar epithelial cells of ectopic endometrium were damaged or exfoliated and the interstitial cells were loose， the content of TNF-α in each dose group of Wenjingtang was significantly decreased， the content of IL-1β and TGF-β1 in the medium dose and high dose groups of Wenjingtang was significantly decreased （P<0.05，P<0.01）， the expression of HIF-1α mRNA and protein were significantly decreased in all Wenjingtang groups （P<0.05，P<0.01）. The mitochondria of ectopic endometrium were obviously swollen， the crest was broken or even disappeared， some of the mitochondria were vacuolar degeneration and the outer membrane was ruptured. Conclusion:Wenjingtang has a good therapeutic effect on experimental EMs in rats， and the mechanism is related to reducing the expression of HIF-1α， improving hypoxia in ectopic lesions and inducing mitochondrial damage in ectopic endometrium.

Effects of active components in compatible solution of Sparganii Rhizoma-Curcumae Rhizoma on pelvic adhesion in rats with chronic pelvic inflammatory disease / 中成药

AIM To study the influence and action mechanism of effective components in compatible solution of Sparganii Rhizoma-Curcumae Rhizoma on pelvic adhesions of model rats with chronic pelvic inflammatory disease.METHODS Laboratory rats were randomly assigned into six groups,a sham-operated group,a model group,a positive Fukeqianjin tablets group,compatible solution groups (40 g/kg high-dose group,20 g/kg medi um-dose group,and 10 g/kg low-dose group).Except rats of the sham-operated group,those in the other groups were induced into models of chronic pelvic inflammatory disease by mechanical bacterial strain implantation,after which a 20 d corresponding drug administration by oral gavage for each group was initiated.After completion of the last drug administration,Verco criterion was taken as the reference to evaluate pelvic adhesions,HE staining was performed on uterine tissues for pathological scoring,ELASA method was applied to measuring serum IL-1β and TNF-α levels,and Western blot method was used to detect the expression levels of proteins FGF-2 and IGF-1 in uterine tissues.RESULTS Compared with the model group,the high-dose compatible solution group had a much sharper fall in Verco scores (P ＜ 0.05),and remarkably decreased serum IL-1β and TNF-α levels (P ＜ 0.01);both high-dose and medium-dose groups manifested with notably alleviated degenerative necrosis and lowered scores of chronic inflammation infiltration and epithelial hyperproliferation (P ＜ 0.01 or P ＜ 0.05),and a distinct reduction in the expression of proteins FGF-2 and IGF-1 (P ＜ 0.05,P ＜ 0.01).CONCLUSION The significant improvement of the chronic pelvic inflammatory disease in model rats due to the effective components in the compatibile solution of Sparganii Rhizoma-Curcumae Rhizoma may be associated with inhibited release of inflammatory mediators IL-1β and TNF-α,and the reduced expression of proteins FGF-2 and IGF-1 as well.

Effect of Paeonia lactiflora formula granule against the damage of LO2 cells induced by carbon tetrachloride / 解放军医学杂志

Objective To investigate the effect of Paeonia lactiflora formula granule (PLFG) against the damage of LO2 cells induced by carbon tetrachloride (CCl4) and explore its mechanism. Methods LO2 cells were divided into control group, CCl4 model group, PLFG (1, 5, 10mg/L) groups and Vit E (50mmol/L) group. LO2 cells of PLFG groups and Vit E group were pretreated by drugs for 24 hours, then except control group, other groups were treated by 10mmol/L CCl4 for 6 hours, which induced hepatic cell damage, the alanine aminotransferase (ALT) and aspartate transaminase (AST) content of the cell culture supernatant were measured, the survival rates of LO2 cells were analyzed by MTT method. LO2 cells were divided into control group, CCl4 model group, PLFG (10mg/L) group and Vit E (50mmol/L) group, cell loss, changes in nuclear size and morphology, DNA content, mitochondrial membrane potential (MMP), cell permeability changes, and cytochrome C release were measured simultaneously by high content analysis (HCA). Results Compared with CCl4 model group, PLFG and Vit E pretreatment could obviously decrease the level of ALT and AST (P<0.05 or P<0.01), especially in the 10mg/L PLFG group and Vit E group. PLFG (1, 5, 10mg/L) and Vit E could also significantly weaken the decrease of LO2 cell viability, which were induced by CCl4 treatment, the results showed in dose-dependent to some extents (P<0.05 or P<0.01). Meanwhile, treatment with 10mg/L PLFG and Vit E could significantly increase the level of MMP (P<0.01), prevent cytochrome C release (P<0.01), decrease the membrane permeability (P<0.01), increase nuclear size (P<0.01), decrease total nuclear intensity (P<0.01) and increase the cell count (P<0.05). Conclusion PLFG may act an obvious protective effect against the liver injury induced by CCl4, and it might be due to protecting the integrity of mitochondria membrane, decreasing cell membrane permeability and inhibiting the apoptosis of LO2 cells.