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.

Modified Wenjingtang Affects Caspase-8， MMP-9， N-cadherin and E-cadherin in Local Microenvironment of EM Rats with Kidney Deficiency and Blood Stasis / 中国实验方剂学杂志

Objective:To investigate the therapeutic effect and mechanism of modified Wenjingtang on endometriosis （EM） rats with kidney deficiency and blood stasis. Method:Healthy non-pregnant female Sprague-Dawley （SD） rats of SPF grade were randomly divided into the blank group and experimental group. After being modeled via soaking in ice water and subcutaneous injection of epinephrine hydrochloride， the ones in the experimental group were further divided into the sham operation group and EM model group， with the former only undergoing laparotomy and the latter further receiving autologous endometrial transplant for triggering EM. The successfully modeled rats with EM due to kidney deficiency and blood stasis were randomized into the positive drug （danazol， 63 mg·kg^-1） group and low- （5 g·kg^-1）， medium- （10 g·kg^-1）， and high-dose （20 g·kg^-1） modified Wenjingtang groups. The corresponding drugs were administered by gavage， once per day， for four weeks. Then the ectopic and eutopic endometrial tissues were stained with hematoxylin-eosin （HE） to observe the histopathological changes. The protein and mRNA expression levels of cysteinyl aspartate-specific proteinase-8 （Caspase-8）， matrix metalloproteinase-9 （MMP-9）， N-cadherin， and E-cadherin were detected by immunohistochemistry （IHC）， Western blotting， and real-time polymerase chain reaction （Real-time PCR）， respectively. Result:The IHC and Western blot revealed that the protein expression levels of MMP-9 and N-cadherin in eutopic and ectopic endometrial tissues of the model group were significantly increased as compared with those in the sham operation group （<italic>P</italic><0.01）， while the levels of Caspase-8 and E-cadherin was significantly decreased （<italic>P</italic><0.01）. Compared with the model group， the danazol and low-， medium-， and high-dose modified Wenjingtang groups exhibited obviously down-regulated MMP-9 and N-cadherin protein expression in eutopic and ectopic endometrial tissues （<italic>P</italic><0.05，<italic>P</italic><0.01）， but up-regulated Caspase-8 and E-cadherin （<italic>P</italic><0.05， <italic>P</italic><0.01）. Real-time PCR uncovered that the mRNA expression levels of MMP-9 and N-cadherin in eutopic and ectopic endometrial tissues of the model group were significantly elevated as compared with those in the sham operation group （<italic>P</italic><0.01）， whereas the levels of Caspase-8 and E-cadherin significantly declined （<italic>P</italic><0.01）. The comparison with the eutopic endometrial tissue in the model group showed that the mRNA expression levels of MMP-9 and N-cadherin in the danazol group and high- and medium-dose modified Wenjingtang groups were significantly down-regulated， while those of Caspase-8 and E-cadherin were significantly up-regulated （<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:Modified Wenjingtang alleviates the immunosuppression and blocks the angiogenesis in EM rats with kidney deficiency and blood stasis syndrome by regulating the expression of such cytokines as Caspase-8， MMP-9， N-cadherin， and E-cadherin， thus exerting the therapeutic effect against EM. The above-mentioned micro-indicators are potential markers reflecting the disease （EM）， syndrome （kidney deficiency and blood stasis）， and pathological mechanisms （immunosuppression and angiogenesis）.

Effect of Modifide Wenjingtang on JAK2/STAT3 Signal Pathway and Downstream Factors Protein Expression in Endometriosis Rats / 中国实验方剂学杂志

Objective:To investigate the therapeutic effect and mechanism of modified Wenjingtang on endometriosis （EM） rats with kidney deficiency and blood stasis. Method:The 10 from 105 SPF female healthy SD rats were randomly selected as the blank group. The rest constructed the rat model of kidney deficiency and blood stasis by compound factorial method. After the model was successfully established， 10 rats were randomly selected as the sham operation group， with only laparotomy and no intima suture， and the remaining rats were established with EM kidney deficiency and blood stasis type by autologous intimal transplantation. Fifty rats which were randomly selected from 56 successful rats were treated with the modified Wenjingtang （5，10，20 g·kg^-1） and danazol group（63 mg·kg^-1）， 1 time daily ， for 4 weeks. The endometrial tissues of each group were stained with hematoxylin eosin （HE） to observe the histopathology. The levels of inflammatory factors interleukin-10 （IL-10） and interleukin-17 （IL-17） in serum supernatant were detected by enzyme linked immunosorbent assay （ELISA）. Measuring the length（D₁），width （D₂） and height （D₃） of the heterotopic foci in each group before and after treatment. Then calculating the volume of them. The expression of tyrosine kinase 2（JAK2），transcription factor 3 （STAT3），phosphorylation transcription factor 3 （p-STAT3）， vascular endothelial growth factor （VEGF）， tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>） and thrombospondin-1 （TSP-1） were detected by immunohistochemistry （IHC）. The expression of VEGF，TNF-<italic>α</italic> and TSP-1 was detected by Western blot. Result:Microscopic pathological observation showed that the endometrial glandular cells of the blank group were arranged in order， and the glandular and stromal cells grew well， compared with the blank group， the endometrial structure of the model group was complete， showing a cavity like or annular closed structure， with cyst formation， and the epithelium was cubic or columnar epithelium， most of the epithelial cells had secretion， the stroma was dense， and the matrix showed a little fibrosis There were a few glands and inflammatory cell infiltration. Compared with the blank group， the content of IL-10 in serum of model group was significantly decreased （<italic>P</italic><0.01）， and the content of IL-17 was significantly increased （<italic>P</italic><0.01）， the protein expression of JAK2， STAT3，p-STAT3， VEGF， TNF-<italic>α</italic> in endometrial tissue of model group was significantly increased （<italic>P</italic><0.05）， and the expression of TSP-1 protein was significantly decreased （<italic>P</italic><0.05）. Compared with the model group， the serum IL-10 content of rats in modified Wenjingtang treatment group increased significantly （<italic>P</italic><0.01）， the IL-17 content decreased significantly <italic>（P</italic><0.01）， and the volume of ectopic foci decreased significantly （<italic>P</italic><0.01）. While the level of JAK2，STAT3，p-STAT3，TNF-<italic>α</italic>，VEGF protein in intimal tissue of modified Wenjingtang high and middle dose group decreased significantly （<italic>P</italic><0.05） and the level of TSP-1 protein increased significantly （<italic>P</italic><0.05）. Conclusion:Modified Wenjingtang can inhibit the invasion of ectopic foci in EM rats with kidney deficiency and blood stasis， the mechanism may be related to the intervention of immune barrier and block angiogenesis function mediated by JAK2/STAT3 signaling pathway activation.

Analysis of Specific Chromatogram of Wenjingtang Reference Sample / 中国实验方剂学杂志

Objective:To control the quality of the reference sample of Wenjingtang by establishing the specific chromatograms. Method:On the basis of analyzing 15 batches of Wenjingtang freeze-dried powder samples， a high performance liquid chromatography （HPLC） specific chromatogram analysis method of Wenjingtang was established. The system adaptability was investigated and the retention time， relative retention value and deviation caused by different chromatographic columns and instruments were calculated by using the same brand of chromatographic columns， four different brands of chromatographic columns and instruments from three different manufacturers. The precision， repeatability and stability of this method was further completed. The possible chemical components of the freeze-dried powders were speculated and identified by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry （UPLC-Q-TOF-MS<italic>ⁿ</italic>）. Chromatographic separation was performed on ACQUITY UPLC BEH C₁₈ column （2.1 mm×100 mm， 1.7 μm） with acetonitrile （A）-0.1% formic acid aqueous solution （B） as mobile phase for gradient elution （0-2.8 min， 10%A； 2.8-8.0 min， 10%-18%A； 8.0-12.2 min， 18%-25%A； 12.2-15.3 min， 25%-40%A； 15.3-17.4 min， 40%A； 17.4-20.5 min， 40%-90%A）， and column temperature was set at 30 ℃ with flow rate of 0.4 mL·min^-1. Mass spectrometry was performed on electrospray ionization， data were collected under positive and negative ion modes， and the detection range was <italic>m</italic>/<italic>z</italic> 50-1 600. Result:Ten characteristic peaks were selected as the distinguishing features in this specific chromatograms， and eight of them were identified by comparing with the reference standards， including paeoniflorin （peak 1）， liquiritin apioside （peak 2）， liquiritin （peak 3）， ferulic acid （peak 4）， iquiritigenin （peak 6）， cinnamaldehyde （peak 8）， paeonol （peak 9）and glycyrrhizic acid （peak 10）. By mass spectrometry analysis， 30 compounds were identified， and the source of medicinal materials were assigned. It mainly contained triterpenoid saponins and flavonoids from Glycyrrhizae Radix et Rhizoma， ginsenosides from Ginseng Radix et Rhizoma， monoterpenoid glycosides and tannins from Paeoniae Radix Alba， steroids in Achyranthis Bidentatae Radix， phenolic acids in Angelicae Sinensis Radix. Conclusion:The established characteristic chromatographic analysis method of Wenjingtang is simple， stable and repeatable. The chemical composition of the freeze-dried powder of Wenjingtang is basically defined by mass spectrometry identification and source attribution， which can provide reference for the development and quality control of Wenjingtang in the future.

Clinical Efficacy of Magui Wenjingtang Combined with Shoulder Joint Loosening Operation in Treating Periarthritis of Shoulder with Syndrome of Blood Deficiency and Cold Coagulation / 中国实验方剂学杂志

Objective::To explore the clinical efficacy of Magui Wenjingtang combined with joint mobilization in the treatment of periarthritis of the shoulder and the effect on hemodynamics and serum pain media. Method::A total of 120 patients with scapulohumeral periarthritis were enrolled in Tianjin Ninghe Hospital from July 2016 to July 2018.All cases were divided into control group and observation group, with 60 patients in each group. The control group received shoulder joint loosening operation (qd), and celecoxib capsules for oral administration. The observation group was treated with Magui Wenjingtang in addition to the shoulder joint loosening operation, 1 dose/d, 2 times/d. After 4 weeks of continuous treatment, the subjects were compared for the subjective pain level McGill Pain Questionnaire (SF-MPQ) score, shoulder mobility hemodynamic changes, and serum prostaglandin E2 (PGE2), substance P (SP), serotonin (5-HT) level and clinical efficacy. Result::Compared with before treatment, visual pain score (VAS), current pain status (PPI), and pain rating index (PRI) scores of the two groups were significantly lower after 4 weeks of treatment, and those of the observation group was significantly lower than the control group (P<0.01). The range of shoulder joint activity was significantly higher than that of the control group (P<0.01), erythrocyte sedimentation rate (ESR), fibrinogen (Fb), whole blood viscosity and serum were compared between the two groups after 4 weeks of treatment and before treatment. The levels of PGE2, SP and 5-HT were significantly lower (P<0.01), and the observation group was significantly better than the control group (P<0.01). The total effective rate of the observation group was 96.61%(57/59), which was significantly higher than 82.76% (48/58) of the control group(χ2=4.685, P<0.05). Conclusion::Magui Wenjingtang combined with shoulder joint loosening operation in treating periarthritis of shoulder with syndrome of blood deficiency and cold coagulation can effectively improve the pain and shoulder joint mobility, increase the curative effect, and improve the hemodynamics, and down-regulatie pain mediator of PGE2, SP, 5-HT, which may be related to the curative effect.

Effect of Wenjingtang on Levels of Serum Platelet Activating Factor and β-endorphins in Primary Dysmenorrhea Patients with Cold Stagnation and Blood Stasis Syndrome / 中国实验方剂学杂志

Objective::To explore the effect of Wenjingtang on the levels of serum platelet activating factor (PAF) and β-endorphins in primary dysmenorrhea patients with cold stagnation and blood stasis syndrome. Method::The 120 cases of dysmenorrhea treated at Second Affiliated Hospital, Heilongjiang University of Chinese Medicine from March 2016 to December 2017 were selected as the observation subjects, and divided into control group and observation group according to the random number table method, with 60 cases in each group. The control group was treated with ibuprofen capsules, while the observation group was treated with Wenjingtang and ibuprofen capsules. Dysmenorrhea symptom scores, clinical efficacy, recurrence condition and serum levels of platelet activating factor and β-endorphins were compared before and after treatment. Result::After treatment, the dysmenorrhea symptom scores decreased significantly in both groups, and the scores of patients in observation group were lower than those in control group (P<0.05). After treatment, the total efficiency in observation group was 95%, which was significantly higher than 83.33% in control group (P<0.05). The level of serum PAF decreased significantly in two groups, and the serum PAF in observation group was significantly decreased compared with control group (P<0.05). The level of serum beta-endorphins in observation group was significantly higher than that of control group (P<0.05). After half a year, the recurrence rate of the observation group was 5%, which was significantly lower than 30% of control group (χ2=12.987, P<0.01). Conclusion::Wenjingtang contributes to the reduction of dysmenorrhea symptoms scores in primary dysmenorrhea patients with cold stagnation and blood stasis syndrome, and can improve the clinical efficacy, reduce the serum PAF level and increase the concentration of β-endorphins, reduce the recurrence rate, improve the quality of life of patients, and it is worth promoting.

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.

Prescription Research and Clinical Application of Wenjingtang / 中国实验方剂学杂志

The prescription research and clinical application of Wenjingtang were summarized in order to provide reference for the formulation of material standard and the development of compound preparation. By systematically sorting out the relevant ancient medical books and modern literature reports， combined with the relevant policy requirements of the development of compound preparations of famous classical formulas， this paper expounded the existing problems and put forward some suggestions. Wenjingtang is composed of nine herbs， which is derived from Complete Effective Prescriptions for Women's Diseases written by CHEN Zi-ming in Song dynasty. The original medicinal plants and medicinal parts of the formula were basically the same as those recorded in the 2020 edition of Chinese Pharmacopoeia， for example， Paeonia lactiflora was the choice of Paeoniae Radix Alba， Cinnamomi Cortex was selected as Guixin， the rhizoma of Curcuma phaeocaulis was selected as Curcumae Rhizoma. It was suggested that raw products should be selected for decoction pieces and processed according to the methods recorded in the 2020 edition of Chinese Pharmacopoeia. According to the dosage of 1 Liang=40 g， the dosages of Angelicae Sinensis Radix， Chuanxiong Rhizoma， Paeoniae Radix Alba， Cinnamomi Cortex， Moutan Cortex and Curcumae Rhizoma were 20 g， the dosages of Ginseng Radix et Rhizoma， Glycyrrhizae Radix et Rhizoma and Achyranthis Bidentatae Radix were 40 g， the total amount of this formula was 240 g. The 9 kinds of decoction pieces were crushed （particle size<6 mm） separately and mixed， each dose was 20 g， 525 mL of water was added， and then decocted to 280 mL. After filtration， warm medicine was taken for once a day. According to ancient books， Wenjingtang has the functions of activating blood circulation， regulating menstruation，warming meridians and dispersing cold. It is mainly used to treat dysmenorrhea， irregular menstruation and other diseases in modern clinical practice. In addition， it has certain curative effect on endometriosis， pelvic inflammatory disease， acne， eczema and other skin diseases. Through the research， the historical evolution and clinical application of Wenjingtang are fully clarified， which can provide research for the later development and application of this famous classical formula.