Objective To explore whether superovulation impairs the process of pregnancy establishment in mice by changing the intrauterine environment. Methods The implantation and pregnancy of superovulated and normal mice were compared. The superovulated mice were subjected to unilateral tubal ligation on day 0. 5 and blastocysts were transplanted to the other uterine horn on day 2. 5. The number of implantation sites of bilateral uterine horn was compared. The differences between preimplantation uteri of superovulated and normal pseudopregnancy mice were compared by tissue sections and high-throughput sequencing. Bioinformatics analysis was performed on the differentially expressed genes in two groups. Results Compared with the control group, the pregnancy rate of mice in the superovulation group decreased significantly. The number of implantation sites in the superovulation group was higher than the control. There was no significant difference in the pregnancy rate of the uterine horn between the control side and the transplanted side of the superovulated mice. The endometrium was thinned and the number of glands was reduced in superovulated pseudopregnancy mice. The gene expression patterns of preimplantation uterus in superovulation pseudopregnancy and normal pseudopregnancy mice were different. There were 1097 significantly differentially expressed genes, including 752 up-regulated genes and 345 down-regulated genes. Bioinformatics analysis showed that differentially expressed genes are mainly involved in biological processes, such as decidualization, response to progesterone, positive regulation of angiogenesis. They were mainly enriched in FoxO signaling pathway, cell cycle pathway and steroid biosynthesis pathway. Conclusion Superovulation impaired the process of establishing pregnancy and altered the gene expression patterns of biomarker of uterine receptivity in mice.

Objective To explore the protective effects and mechanisms of cysteinyl leukotriene 2(CysLT2)receptor antagonist HAMI3379 on cerebral ischemia injury in rats.Methods 30 male SD rats were randomly divided into sham operation group,model group,and HAMI3379 group,with 10 rats in each group.The rats of model group were subjected to middle cerebral artery occlusion(MCAO)to construct the cerebral ischemia injury model,while HAMI3379 group received intraperitoneal injection of HAMI3379(0.2 mg/kg)before and after MCAO 30 min.The rats after cerebral ischemia injury were scored for neurological symptoms.The infarction volume of rats was observed by TTC staining,the activation marker Iba1 of microglia was detected by immunofluorescence staining,the mRNA level of M1/M2 polarized phenotype molecules of microglia was detected by Real-time PCR,the number of neurons was observed by NeuN staining,and neuronal degeneration was observed by Fluoro-Jade B staining.Western blotting assay was used to detect the expression of CysLT2 protein and nuclear factors κB-related protein Cα(PKCα),IκBα,p65 and p50 proteins in brain tissue.Results Compared with sham operation group,the neurological symptom score and cerebral infarction volume of model group were significantly increased(P<0.05).Compared with model group,the neurological symptom score and cerebral infarction volume of HAMI3379 group were significantly decreased(P<0.05).The results of immunofluorescence staining showed that the expression of microglia activation marker Iba1 was increased in brain tissue of rats after cerebral ischemia injury(P<0.05).Compared with sham operation group,mRNA expression of M1 polarized molecules(CD86,IL-1β,TNF-α)and M2 polarized molecules(CD206,TGF-β,IL-10)were significantly increased in the ischemic central brain tissue of model group(P<0.05).Compared with model group,the expression of M1 polarized molecules in HAMI3379 group was significantly downregulated(P<0.05),while the expression of M2 polarized molecules was significantly upregulated(P<0.05).Compared with sham operation group,the expressions of PKCα,IκBα,p65 and p50 in brain tissue of model group were significantly up-regulated(P<0.05);compared with model group,the expressions of PKCα,IκBα,p65,and p50 in HAMI3379 group were significantly down-regulated(P<0.05).The NeuN staining results showed that the number of neurons in the brain tissue of model group was decreased when compared with sham operation group(P<0.05),while the number of degenerated neurons was increased(P<0.05).Compared with model group,the number of neurons in HAMI3379 group was increased(P<0.05),while the number of degenerated neurons was decreased(P<0.05).Conclusions CysLT2 receptor antagonist HAMI3379 may regulate PKCα/IκBα/NF-κB signaling pathway,inhibits M1 polarization activation of microglia and promotes its transition to M2 polarization,inhibits neuronal degeneration,and plays a neuroprotective role.

UPLC-Q-Exactive-MS/MS and network pharmacology were employed to preliminarily study the active components and mechanism of Jinwugutong Capsules in the treatment of osteoporosis. Firstly, UPLC-Q-Exactive-MS/MS was employed to characterize the chemical components of Jinwugutong Capsules, and network pharmacology was employed to establish the "drug-component-target-pathway-disease" network. The key targets and main active components were thus obtained. Secondly, AutoDock was used for the molecular docking between the main active components and key targets. Finally, the animal model of osteoporosis was established, and the effect of Jinwugutong Capsules on the expression of key targets including RAC-alpha serine/threonine-protein kinase(AKT1), albumin(ALB), and tumor necrosis factor-alpha(TNF-α) was determined by enzyme-linked immunosorbent assay(ELISA). A total of 59 chemical components were identified from Jinwugutong Capsules, among which coryfolin, 8-prenylnaringenin, demethoxycurcumin, isobavachin, and genistein may be the main active components of Jinwugutong Capsules in treating osteoporosis. The topological analysis of the protein-protein interaction(PPI) network revealed 10 core targets such as AKT1, ALB, catenin beta 1(CTNNB1), TNF, and epidermal growth factor receptor(EGFR). The Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment showed that Jinwugutong Capsules mainly exerted the therapeutic effect by regulating the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT) signaling pathway, neuroactive ligand-receptor interaction, mitogen-activated protein kinase(MAPK) signaling pathway, Rap1 signaling pathway and so on. Molecular docking showed that the main active components of Jinwugutong Capsules well bound to the key targets. ELISA results showed that Jinwugutong Capsules down-regulated the protein levels of AKT1 and TNF-α and up-regulated the protein level of ALB, which preliminarily verified the reliability of network pharmacology. This study indicates that Jinwugutong Capsules may play a role in the treatment of osteoporosis through multiple components, targets, and pathways, which can provide reference for the further research.
Animals ; Tumor Necrosis Factor-alpha/genetics* ; Network Pharmacology ; Capsules ; Molecular Docking Simulation ; Phosphatidylinositol 3-Kinases ; Reproducibility of Results ; Tandem Mass Spectrometry

Nasopharyngeal carcinoma (NPC) is the third most common malignancy with a high recurrence and metastasis rate in South China. Natural compounds extracted from traditional Chinese herbal medicines have been developed and utilized for the treatment of a variety of cancers with modest properties and slight side effects. Maackiain (MA) is a type of flavonoid that was first isolated from leguminous plants, and it has been reported to relieve various nervous system disorders and exert anti-allergic as well as anti-inflammatory effects. In this study, we demonstrated that MA inhibited proliferation, arrested cell cycle and induced apoptosis in nasopharyngeal carcinoma CNE1 and CNE2 cells in vitro and in vivo. The expression of the related proteins associated with these processes were consistent with the above effects. Moreover, transcriptome sequencing and subsequent Western blot experiments revealed that inhibition of the MAPK/Ras pathway may be responsible to the anti-tumor effect of MA on NPC cells. Therefore, the effects of MA and an activator of this pathway, tertiary butylhydroquinone (TBHQ), alone or combination, were investigated. The results showed TBHQ neutralized the inhibitory effects of MA. These data suggest that MA exerts its anti-tumor effect by inhibiting the MAPK/Ras signaling pathway and it has the potential to become a treatment for patients with NPC.
Humans ; Nasopharyngeal Carcinoma/pathology* ; Cell Line, Tumor ; Cell Proliferation ; Apoptosis ; Signal Transduction ; Nasopharyngeal Neoplasms/pathology*

This study aims to evaluate the quality consistency of Saposhnikoviae Radix based on carbohydrates, and explore the potential of carbohydrates as the internal quality control indicators of Saposhnikoviae Radix. The total polysaccharides were quantified by UV-Vis spectrophotometry and the molecular weight range of the polysaccharides was determined by high performance gel-permeation chromatography-evaporative light scattering detection(HPGPC-ELSD). The monosaccharides in polysaccharides and the free monosaccharides were quantified by high performance liquid chromatography-UV detection(HPLC-UV), and the oligosaccharides and fructose were quantified by high performance liquid chromatography-evaporative light scattering detection(HPLC-ELSD). The carbohydrate-based quality of Saposhnikoviae Radix was compared among 45 batches of commercial samples and 13 batches of self-collected samples. The results showed that the molecular weight distribution, monosaccharide composition, oligosaccharide, and free monosaccharide composition were similar in the 58 batches of samples. The average content of total polysaccharides, oligosaccharides, and total free monosaccharides in commercial samples were 39.66, 148.79, and 68.62 mg·g~(-1), respectively. The content showed significant differences among batches, with the highest differences of 3.51, 1.75, and 2.58 times, respectively. The RSD of the relative ratios of monosaccharides in the polysaccharides in commercial samples reached 28%-45%. The average content of total polysaccharides, oligosaccharides, and total free monosaccharides in self-collected samples were 68.07, 145.76, and 42.04 mg·g~(-1), respectively, with the inter-region differences of 2.88, 1.88, and 1.07 times, respectively. The RSD of the relative ratios of monosaccharides in polysaccharides in self-collected samples ranged from 8.2% to 59%. The total polysaccharides and total free monosaccharides in self-collected samples were 1.72 times higher and 1.63 times lower, respectively, than those in commercial samples. The content of oligosaccharides was similar between self-collected samples and commercial samples. To sum up, carbohydrates are one of the material bases for the internal quality consistency of Saposhnikoviae Radix. The qualitative characteristics of polysaccharides and the quantitative characteristics of polysaccharides and oligosaccharides are related to the origin of medicinal materials. Moreover, the quantitative characteristics of polysaccharides and free monosaccharides may be related to the storage conditions. Carbohydrates are potential indicators for the quality control of Saposhnikoviae Radix and deserve attention.

OBJECTIVE: To observe the clinical effect of high suspension and low incision (HSLI) surgery on mixed haemorrhoids, compared with Milligan-Morgan haemorrhoidectomy. METHODS: A multi-centre, randomized, single-blind, non-inferiority clinical trial was performed. Participants with mixed haemorrhoids from Xiyuan Hospital of China Academy of Chinese Medical Sciences, Beijing Rectum Hospital, Air Force Medical Center of People's Liberation Army of China, and Puyang Hospital of Traditional Chinese Medicine were enrolled from September 2016 to March 2018. By using a blocked randomization scheme, participants were assigned to two groups. The experimental group was treated with HSLI, while the control group was treated with Milligan-Morgan haemorrhoidectomy. The primary outcome was the clinical effect evaluated at 12 weeks after operation. The secondary outcomes included the number of haemorrhoids treated during the operation, pain scores, use of analgesics, postoperative oedema, wound healing, incidence of anal stenosis, anorectal manometry after operation, as well as surgical duration, length of stay and total hospitalization expenses. A safety evaluation was also conducted. RESULTS: In total, 246 eligible participants were enrolled, with 123 cases in each group. There was no significant difference in the clinical effect between the two groups (100.00% vs. 99.19%, P>0.05). Compared with the control group, the number of external haemorrhoids treated during the operation and the pain scores after operation were significantly reduced in the experimental group (P<0.05 or P<0.01); the patient number with wound healing at 2 weeks after operation and the functional length of anal canal at 12 weeks after operation were significantly increased in the experimental group (P<0.05). There was no significant difference in the incidence of anal stenosis, the numbers of patients using analgesics and patients with postoperative oedema between the two groups after operation (P>0.05). The surgical duration and length of stay in the experimental group were significantly longer than those in the control group, and the total hospitalization expense was significantly higher than that in the control group (all P<0.05). No adverse events were reported in either group during the whole trial or follow-up period. CONCLUSION HSLI had the advantages of preserving the skin of anal canal completely, alleviating postsurgical pain and promoting rapid recovery after operation. (Registration No. ChiCTR1900022883).

Objective:To prepare biomimetic tissue engineering scaffolds of gelatin/sodium alginate/laponite composite hydrogel loaded with BMSCs by 3D biological printing technique，and explore the osteogenic effect of 3D printing on hydrogel scaffolds containing bone marrow mesenchymal stem cells（BMSCs）.Methods:BMSCs were routinely extracted and identified by flow cytometry. Gelatin，sodium alginate and laponite were mixed and then BMSCs were added to prepare cell-containing composite hydrogel scaffolds using 3D bioprinting. Non-printed scaffolds containing cells were prepared by injection molding method. In vitro，the prepared scaffolds were divided into the printing group with cells and non-printing group with cells according to whether they were printed，with 12 samples per group. Another simple cell culture group was set as control. Then，the internal structure of the composite hydrogel was observed by scanning electron microscope，and the expansion rate and water content of the scaffolds were measured by freeze-drying method. At day 3 after culture，the growth status of BMSCs was observed by phalloidine staining. cell counting kit（CCK）-8 assay was used to detect cell activity in scaffolds at days 1，3，and 7 after culture and RT-PCR to detect the expression of osteogenesis related genes Osterix，osteocalcin（OCN）and collagen I at days 7 and 14 ofter culture. In vivo，four groups were set according to printing or not and whether containing cells or not：printing implant group with cells，non-printing implant group with cells，printing implant group without cells and non-printing implant group without cells，with 9 samples per group. Scaffolds in four groups were implanted to the posterior gluteal muscle pouches（random on left or right）of 36 8-week-old SD rats，respectively. The samples were taken X-ray images at 2，4 and 8 weeks after operation，respectively. The osteogenic differentiation of tissues at 8 weeks was observed by HE and Masson staining. Results:The flow cytometry showed that the cells were BMSCs. Internal pores of hydrogels were obvious，and cells stretched freely in the pores. Differences of the swelling rate and water content were not statistically significant between printing group with cells［（1，039.37±30.66）%，（91.21±0.26）%］and non-printing group with cells［（1，032.38±35.05）%，（91.16±0.28）%］（ P>0.05）. At day 3 after culture in vitro，the cells grew well in the hydrogel. After culturing for 1 day in vitro，there was no significant difference in absorbance between printing group with cells and non-printing group with cells（ P>0.05）. At day 3 after culture，there was no significant difference in absorbance between printing group with cells and non-printing group with cells，but both groups showed a higher level than simple cell culture group（ P<0.05）. At day 7 after culture，the absorbance in printing group with cells（2.72±0.17）was higher than that in non-printing group with cells（2.35±0.11），and both of which were higher than that in simple cell culture group（1.95±0.12）（ P<0.05）. At day 7 after culture in vitro，there was no statistically significant difference in the expression of osteogenic differentiation-related genes between printing group with cells and the non-printing group with cells（ P>0.05），but they were all higher than those in simple cell culture group（ P<0.05）. At day 14 after culture in vitro，the expression of osteogenesis-related genes Osterix（1.650±0.095），OCN（2.725±0.091），collagen I（2.024±0.091）in printing group with cells were higher than those in non-printing group with cells（1.369±0.114，2.174±0.198，1.617±0.082，respectively）and those in simple cell culture group（1.031±0.094，1.116±0.092，0.736±0.140，respectively）（ P<0.05）. After implantation for 2 weeks in vivo，with no statistically significant difference in the gray values of X-ray films in each group（ P>0.05）. At weeks 4 and 8 after implantation，the gray values of X-ray films in printing implant group with cells and non-printing implant group with cells were higher than those in printing implant group without cells and non-printing implant group without cells（ P<0.01）. At 8 weeks after implantation，HE staining showed that the scaffolds were degraded in different degrees and immersed with cells，with collagen production seen in Masson staining as well. Conclusions:Composite hydrogel scaffolds can provide a good three-dimensional environment for BMSCs growth. 3D bioprinting can promote the proliferation and osteogenic differentiation of BMSCs in hydrogel scaffolds. In addition，BMSCs-loaded scaffolds can be degraded slowly in vivo with good ectopic osteogenic ability.

Purpose To investigate the expression and clinical significance of free fatty acid receptor 4 (FFAR4) in hepatocellular carcinoma (HCC) . Methods The expression of FFAR4 in HCC tissues and adjacent tissues of HCC patients was confirmed by 102 cases of liver resection and postoperative pathology, and the relationship between FFAR4 expression and clinical data of HCC patients was analyzed. Quantitative realtime PCR (qRT-PCR) and Western blot were used to detect the expression of FFAR4 in 20 pairs of freshly frozen HCC and adjacent tissues,and the related literatures were reviewed. Results The expression rate of FFAR4 in HCC tissues was 64. 7％ (66/102) ,and that in adjacent tissues was 15. 7％ (16/102) . The difference in FFAR4 expression between the two groups was statistically significant (P < 0. 05) . The high expression of FFAR4 in HCC tissues was significantly correlated with tumor vascular invasion (P < 0. 05) ,TNM stage (P < 0. 01) ,and Edmondson classification (P < 0. 05) . qRT-PCR and Western blot showed that the expression of FFAR4 in HCC tissues was significantly higher than that in adjacent tissues. The difference between the two groups was statistically significant (P < 0. 01,P< 0. 05) . Conclusion The expression of FFAR4 is significantly associated with the presence of vascular invasion,TNM staging, and Edmondson grading in HCC. High expression of FFAR4 may be closely related to the severity of HCC patients.

The formation of expert consensus statement is an indispensable part in the process of developing clinical practice guidelines. The Guidelines International Network believes that experts make group decisions for different stages and issues,and the process of gathering expert opinions is the process of reaching the consensus. GRADE system also requires that recommendations should be formed based on expert consensus in consideration of the risk and bias,patients' preferences and values,resources and other factors. At present,the main method for reaching consensus is the formal consensus method. According to the published clinical guidelines,most of them failed to report the specific methods and process of reaching expert consensus. Therefore,it is impossible to obtain an objective evaluation. This phenomenon is more common in the field of clinical practice guidelines of traditional Chinese medicine( TCM). There are two main reasons for this phenomenon. For one thing,the developers of the guidelines neglect the importance of transparency and objectivity in the implementation of expert consensus. For another,they know little about the methods and technical specifications for the formation of expert consensus. To solve them,based on the internationally recognized consensus-building methods,as well as the specific stages in the process of developing clinical practice guidelines of traditional Chinese medicine,it is of great significance to put forward the technical norms for TCM researchers to develop the expert consensus. This guide will provide detailed guidance for forming the expert consensus for TCM clinical practice guideline. This guideline has been approved and published by the Chinese Association of Traditional Chinese Medicine( No. T/CACM 1049-2017).
Consensus ; Humans ; Medicine, Chinese Traditional ; Practice Guidelines as Topic

OBJECTIVE: To study the effects of aerobic exercise combined with chlorella pyrenoidos of disintegrated cell wall on the lipid metabolism in rats with high-fat diet. METHODS: Fifty-five male Wistar rats were subjected to adaptive feeding for 4 days and weight-free swimming training for 3 days, 20 min/d. After eliminating 5 rats that were not suitable for swimming training, the other rats were randomly divided into 5 groups according to their body weight:control group (C group), high fat diet group (H group), high-fat diet + chlorella group(HC group), high fat diet + aerobic exercise group (HM group), high fat diet + chlorella + aerobic exercise group (HMC group), 10 in each group. The HM and HMC group were subjected to 60 min/d swimming training for 6 weeks with non-weight-bearing. Group C were fed regular diet. The other groups were fed with high-fat diet, the rats in group HC and HMC were intragastrically treated with chlorella pyrenoidos of disintegrated cell wall at the dose of 3.9 g/(kg·d), the volume was 5 ml/kg, and the other groups are given equivalent saline. The Lee's index and biochemical indexes of blood and liver were measured after 6 weeks. RESULTS: Compared with group C, Lee's index, serum levels of free fatty acids(FFA), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-c), liver FFA and interleukin-10 (IL-10) were increased significantly (<0.01), the serum level of high-density lipoprotein cholesterol (HDL-c) was decreased significantly (<0.01) in group H. Compared with group H, Lee's index, serum FFA, IL-6, TNF-α, TC, TG, LDL-c, liver FFA and IL-10 were decreased significantly (<0.05 or <0.01), serum level of HDL-c was increased significantly (<0.05 or <0.01) in group HC, HM and HMC. Compared with group HC and HM, Lee's index, serum FFA, IL-6, TNF-α, TC, TG, LDL-c, liver FFA and IL-10 were decreased significantly (<0.05), serum level of HDL-c was increased significantly (<0.05) in group HMC. CONCLUSIONS Aerobic exercise and chlorella pyrenoidos of disintegrated cell wall can improve lipid metabolism in rats with high-fat diet and reduce the lipid toxicity caused by obesity. Joint intervention is more effective than single intervention.
Animals ; Cell Wall ; Chlorella ; Diet, High-Fat ; Lipid Metabolism ; Male ; Physical Conditioning, Animal ; Rats ; Rats, Wistar