Fuzzy identification of bioactive components for different efficacies of rhubarb by the back propagation neural network association analysis of UPLC-Q-TOF/MSE and integrated effects.

BACKGROUND: Rhei Radix et Rhizoma (rhubarb), as one of the typical representatives of multi-effect traditional Chinese medicines (TCMs), has been utilized in the treatment of various diseases due to its multicomponent nature. However, there are few systematic investigations for the corresponding effect of individual components in rhubarb. Hence, we aimed to develop a novel strategy to fuzzily identify bioactive components for different efficacies of rhubarb by the back propagation (BP) neural network association analysis of ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry for every data (UPLC-Q-TOF/MSE) and integrated effects. METHODS: Through applying the fuzzy chemical identification, most components of rhubarb were classified into different chemical groups. Meanwhile the integration effect values of different efficacies can be determined by animal experiment evaluation and multi-attribute comprehensive indexes. Then the BP neural network was employed for association analysis of components and different efficacies by correlating the component contents determined from UPLC-Q-TOF/MSE profiling and the integration effect values. Finally, the effect contribution of one type of components may be totaled to demonstrate the universal and individual characters for different efficacies of rhubarb. RESULTS: It suggested that combined anthraquinones, flavanols and their polymers may be the universal character to the multi-functional properties of rhubarb. Other components contributed to the individuality of rhubarb efficacies, including stilbene glycosides, anthranones and their dimers, free anthraquinones, chromones, gallic acid and gallotannins, butyrylbenzenes and their glycosides. CONCLUSIONS: Our findings demonstrated that the bioactive components for different efficacies of rhubarb were not exactly the same and can be systematically differentiated by the network-oriented strategy. These efforts will advance our knowledge and understanding of the bioactive components in rhubarb and provide scientific evidence to support the expansion of its use in clinical applications and the further development of some products based on this medicinal herb.

Study on changes in pigment composition during the blooming period of safflower based on plant metabolomics and semi-quantitative analysis.

Red and yellow pigments are the major ingredients of safflower, often used to color food and cosmetics. Carthamin was the main component of red pigment and hydroxysafflor yellow A and anhydrosafflower yellow B were representative components of yellow pigment. Plant metabolomics and semi-quantitative analysis were used to analyze the changes of pigment composition during the blooming period, especially these characteristic components. Carthamin, hydroxysafflor yellow A, anhydrosafflower yellow B, and other components were screened out as differential metabolites based on plant metabolomics. Then semi-quantitative analysis was used to quantify these three representative components of pigments. Experimental results showed that the content of pigments has dynamic changes along with flowering, in the early blooming period, yellow pigment accumulated much and red pigment was low in content. In the middle period, the accumulation rate of the yellow pigment slowed down and content was stabilized. In the next step, the content of yellow pigments gradually decreased, and the content of red pigments gradually increased. Later, the level of yellow pigment decreased significantly, and the accumulation rate of red pigment increased significantly. Last, the appearance color of safflower was red, with yellow parts barely visible, and accumulation of red pigment was the highest and of the yellow pigment was the lowest in content.

Studies on blood enrichment and anti-tumor effects of combined Danggui Buxue Decoction, Fe and rhEPO based on colon cancer-related anemia model and gut microbiota modulation.

Colon cancer-related anemia (CCRA) is mainly caused by systemic inflammation, intestinal bleeding, iron deficiency and chemotherapy-induced myelosuppression in colon cancer. However, the best therapeutic schedule and related mechanism on CCRA were still uncertain. Studies on blood enrichment and anti-tumor effects of combined Danggui Buxue Decoction (DBD), Fe and rhEPO based on CCRA and gut microbiota modulation were conducted in this paper. Here, CCRA model was successfully induced by subcutaneous inoculation of CT-26 and i.p. oxaliplatin, rhEPO + DBD high dosage + Fe (EDF) and rhEPO + DBD high dosage (ED) groups had the best blood enrichment effect. Attractively, EDF group also showed antitumor activity. The sequencing results of gut microbiota showed that compared to P group, the relative abundances of Lachnospiraceae and opportunistic pathogen (Odoribacter) in ED and EDF groups were decreased. Interestingly, EDF also decreased the relative abundances of cancer-related bacteria (Helicobacter, Lactococcus, Alloprevotella) and imbalance-inducing bacteria (Escherichia-Shigella and Parabacteroides) and increased the relative abundances of butyrate-producing bacteria (Ruminococcaceae_UCG-014), however, ED showed the opposite effects to EDF, this might be the reason of the smaller tumor volume in EDF group. Our findings proposed the best treatment combination of DBD, rhEPO and Fe in CCRA and provided theoretical basis and literature reference for CCRA-induced intestinal flora disorder and the regulatory mechanism of EDF.

Total Flavonoids of Glycyrrhiza uralensis Alleviates Irinotecan-Induced Colitis via Modification of Gut Microbiota and Fecal Metabolism.

Irinotecan (CPT-11)-induced gastrointestinal toxicity strongly limits its anticancer efficacy. Glycyrrhiza uralensis Fisch., especially flavonoids, has strong anti-inflammatory and immunomodulatory activities. Herein, we investigate the protective effect of the total flavonoids of G. uralensis (TFGU) on CPT-11-induced colitis mice from the perspective of gut microbiota and fecal metabolism. The body weight and colon length of mice were measured. Our results showed that oral administration of TFGU significantly attenuated the loss of body weight and the shortening of colon length induced by CPT-11. The elevated disease activity index and histological score of colon as well as the up-regulated mRNA and protein levels of TNF-α, IL-1ß, and IL-6 in the colonic tissue of CPT-11-treated mice were significantly decreased by TFGU. Meanwhile, TFGU restored the perturbed gut microbial structure and function in CPT-11-treated mice to near normal level. TFGU also effectively reversed the CPT-11-induced fecal metabolic disorders in mice, mainly call backing the hypoxanthine and uric acid in purine metabolism. Spearman's correlation analysis further revealed that Lactobacillus abundance negatively correlated with fecal uric acid concentration, suggesting the pivotal role of gut microbiota in CPT-11-induced colitis. Since uric acid is a ligand of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, TFGU was further validated to inhibit the activation of NLRP3 inflammasome by CPT-11. Our findings suggest TFGU can correct the overall gut microbial dysbiosis and fecal metabolic disorders in the CPT-11-induced colitis mice, underscoring the potential of using dietary G. uralensis as a chemotherapeutic adjuvant.

Quantitative evaluation of Danqi tablet by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry integrated with bioassay.

Danqi tablet composed of the dried roots of Salvia miltiorrhiza and Panax notoginseng is a well-known Chinese patent medicine commonly used for the treatment of cardio-cerebrovascular diseases such as coronary heart disease and myocardial ischemia. Numerous chemical constituents belonging to S. miltiorrhiza and P. notoginseng were detectable in Danqi tablet. Here, we established and validated a rapid and sensitive ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry method for simultaneous quantification of 23 components in Danqi tablet and then successfully applied to assay 12 batches of samples from ten manufacturers. Our results demonstrated that the contents of 23 components in 12 batches of Danqi tablets varied significantly and their quality indeed existed differently based on the principal component analysis. According to the quantitative data and the loading plot of principal component analysis, five abundant compounds in Danqi tablet were selected as characteristic chemical markers possibly responsible for the quality assessment. Among them, salvianolic acid B and ginsenoside Rg1 were further chosen to be combined at 2:5 ratio to evaluate the anti-thrombotic activity on phenylhydrazine-induced zebrafish heart thrombosis model. Expectedly, this component combination increased the heart red blood cells intensity compared with the model group and the median effective concentration was 123.4 µg/mL, suggestive of its well anti-thrombotic effect. This study contributed to the quantitative evaluation of Danqi tablet and indicated the combination of salvianolic acid B and ginsenoside Rg1 may be capable of reflecting the effect of Danqi tablet, thereby providing a reference for further investigations on the improvement of quality control and clinical application of Danqi tablet.

Integration of organ metabolomics and proteomics in exploring the blood enriching mechanism of Danggui Buxue Decoction in hemorrhagic anemia rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Danggui Buxue Decoction (DBD), as a classical Chinese medicine prescription, is composed of Danggui (DG) and Huangqi (HQ) at a ratio of 1:5, and it has been used clinically in treating anemia for hundreds of years. AIM OF THE STUDY: The aim of this study was to explore the treatment mechanisms of DBD in anemia rats from the perspective of thymus and spleen. MATERIALS AND METHODS: In this study, a successful hemorrhagic anemia model was established, and metabolomics (UPLC-QTOF-MS/MS) and proteomics (label-free approach) together with bioinformatics (Gene Ontology analysis and Reactome pathway enrichment), correlation analysis (pearson correlation matrix) and joint pathway analysis (MetaboAnalyst) were employed to discover the underlying mechanisms of DBD. RESULTS: DBD had a significant blood enrichment effect on hemorrhagic anemia rats. Metabolomics and proteomics results showed that DBD regulated a total of 10 metabolites (lysophosphatidylcholines, etc.) and 41 proteins (myeloperoxidase, etc.) in thymus, and 9 metabolites (L-methionine, etc.) and 24 proteins (transferrin, etc.) in spleen. With GO analysis and Reactome pathway enrichment, DBD mainly improved anti-oxidative stress ability of thymocyte and accelerated oxidative phosphorylation to provide ATP for splenocyte. Phenotype key indexes were strongly and positively associated with most of the differential proteins and metabolites, especially nucleosides, amino acids, Fabp4, Decr1 and Ndufs3. 14 pathways in thymus and 9 pathways in spleen were obtained through joint pathway analysis, in addition, the most influential pathway in thymus was arachidonic acid metabolism, while in spleen was the biosynthesis of phenylalanine, tyrosine and tryptophan. Furthermore, DBD was validated to up-regulate Mpo, Hbb and Cp levels and down-regulate Ca2+ level in thymus, as well as up-regulate Fabp4, Ndufs3, Tf, Decr1 and ATP levels in spleen. CONCLUSION: DBD might enhance thymus function mainly by reducing excessive lipid metabolism and intracellular Ca2+ level, and promote ATP production in spleen to provide energy.

Analysis and evaluation of nucleosides, nucleobases, and amino acids in safflower from different regions based on ultra high performance liquid chromatography coupled with triple-quadrupole linear ion-trap tandem mass spectrometry.

Safflower has both medicinal and edible values but research on its nutrient composition is still lacking. This study was established for the quantitative determination of 28 nucleosides, nucleobases, and amino acids based on the ultra-performance liquid chromatography coupled with triple-quadrupole linear ion-trap tandem mass spectrometry. Analysis of 30 batches of safflower from different producing areas indicated that the contents of l-proline, l-asparagine, l(+)-arginine, l-serine, l-histidine, uracil, guanosine, and uridine was high in safflower. Principle component analysis and cluster analysis found that samples from different regions could be distinguished well, and samples from the same area could be clustered into one class, different geographical environments may cause the differences of nucleosides, nucleobases, and amino acids in safflower. The analysis of principal component analysis, cluster analysis, and counter propagation artificial neural network show similar results. Then the content of nucleosides, nucleobases, and essential amino acids were compared, and found that the content in safflower from Gansu was higher than those from other regions, and there was a little difference between the samples from Xinjiang, Sichuan, and Yunnan. This research revealed the composition of nucleosides, nucleobases, and amino acids in safflower, and provided a theoretical basis for utilization of safflower.

Deciphering the Active Compounds and Mechanisms of Qixuehe Capsule on Qi Stagnation and Blood Stasis Syndrome: A Network Pharmacology Study.

BACKGROUND: Qixuehe capsule (QXH), a Chinese patent medicine, has been demonstrated to be effective in the treatment of menstrual disorders. In traditional Chinese medicine (TCM) theory, qi stagnation and blood stasis syndrome (QS-BSS) is the main syndrome type of menstrual disorders. However, the pharmacodynamic effect of QXH in treating QS-BSS is not clear, and the main active compounds and underlying mechanisms remain unknown. METHODS: A rat model of QS-BSS was established to evaluate the pharmacodynamic effect of QXH. Thereafter, a network pharmacology approach was performed to decipher the active compounds and underlying mechanisms of QXH. RESULTS: QXH could significantly reduce the rising whole blood viscosity (WBV) and plasma viscosity (PV) but also normalize prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (FIB) content in QS-BSS rats. Based on partial least-squares-discriminant analysis (PLS-DA), the low-dose QXH-intervened (QXH-L) and the high-dose QXH-intervened (QXH-H) groups seemed the most effective by calculating the relative distance to normality. Through network pharmacology, QXH may improve hemorheological abnormality mainly via 185 compounds-51 targets-28 pathways, whereas 184 compounds-68 targets-28 pathways were associated with QXH in improving coagulopathy. Subsequently, 25 active compounds of QXH were verified by UPLC-Q/TOF-MS. Furthermore, 174 active compounds of QXH were shared in improving hemorheological abnormality and coagulopathy in QS-BSS, each of which can act on multiple targets to be mainly involved in complement and coagulation cascades, leukocyte transendothelial migration, PPAR signaling pathway, VEGF signaling pathway, and arachidonic acid metabolism. The attribution of active compounds indicated that Angelicae Sinensis Radix (DG), Paeoniae Radix Rubra (CS), Carthami Flos (HH), Persicae Semen (TR), and Corydalis Rhizoma (YHS) were the vital herbs of QXH in treating QS-BSS. CONCLUSION: QXH can improve the hemorheology abnormality and coagulopathy of QS-BSS, which may result from the synergy of multiple compounds, targets, and pathways.

Regulation of serum lipidomics and amino acid profiles of rats with acute myocardial ischemia by Salvia miltiorrhiza and Panax notoginseng herb pair.

BACKGROUND: Salvia miltiorrhiza and Panax notoginseng herb pair (DQ) has been widely used in traditional Chinese medicine for a long history to prevent and treat the coronary heart disease. However, its protective mechanisms against myocardial ischemia during coronary heart disease remain not well-understood. PURPOSE: In this study, we aimed to explore the protective mechanisms of DQ on myocardial ischemia from the perspective of serum lipidomics and amino acids (AAs). METHODS: Rats were orally administrated with low-dose DQ (L-DQ, 0.24 g/kg) and high-dose DQ (H-DQ, 0.96 g/kg) for two weeks and subcutaneously injected with isoproterenol (ISO, 65 mg/kg) for two consecutive days (13th and 14th days) to induce acute myocardial ischemia (AMI). Heart histopathology and serum biochemical indices were examined. The specifically altered serum lipid metabolites were profiled via lipidomics approach, while serum AA profiles were analyzed using UHPLC-TQ-MS/MS. RESULTS: Cardiac marker enzymes (CK, CK-MB, LDH and cTn-I) were significantly upregulated in AMI rats with some of which significantly dropped to normal level in L- and H-DQ groups. Serum TC, TG, HDL, LDL, VLDL and FFA were improved in AMI rats treatment with L- and H-DQ. Further, the PCA based on lipidomics showed serum lipid metabolites in L- and H-DQ groups were closer to control group than that in model group. Compared with model group, H-DQ pretreatment significantly reduced SM (d34:1) and CE (20:4), and increased FA (20:5), PC (26:1), TG (56:9), TG (54:7), MG (17:0), Cer (d32:0) and Cer (d34:0), whereas L-DQ significantly alleviated the perturbed levels of CE (20:4), FA (20:5), MG (17:0), and SM (d34:1). Moreover, there was a significant increment for leucine, isoleucine, valine, phenylalanine, lysine and glutamate but a significant reduction for tryptophan in the serum of rats in model group as compared to control group. Intriguingly, H-DQ could significantly decrease the levels of glutamate, lysine, isoleucine, and BCAAs (the sum of leucine, isoleucine and valine) after AMI, while L-DQ had no significant effects on the above altered AAs. The Western blotting results implied that H-DQ could promote the myocardial BCAA catabolism in AMI rats by activation of BCKDHA, whereas by inhibition of BCKDHK. CONCLUSION: This study presents evidence for the therapeutic effects of DQ on AMI injury, in part, via co-regulating lipid and AA metabolisms.

An integrated metabolomics strategy to reveal dose-effect relationship and therapeutic mechanisms of different efficacy of rhubarb in constipation rats.

The ambiguity of dose-effect relationship of many traditional Chinese medicines (TCMs) has always influenced their rational use in TCM clinic. Rhubarb, a preferred representative of cathartic TCM, is currently widely used that results in a diversity of its dosage. The aim of this study was to use an integrated metabolomics strategy to simultaneously reveal dose-effect relationship and therapeutic mechanisms of different efficacy of rhubarb in constipation rats. Six doses of rhubarb (0.135, 0.27, 0.81, 1.35, 4.05, and 8.1 g/kg) were examined to elucidate the laxative and fire-purging effects by pathological sections and UPLC-Q-TOF/MSE. The results showed that there existed serious lesions in the stomach and colon of model rats. And conditions were basically improved to some extent in rhubarb-treated groups. Through relative distance calculation based on metabolomics score plots, it suggested that the effective dose threshold (EC20-EC80 range) of rhubarb was from 0.31 to 4.5 g/kg (corresponding to 3.44-50.00 g in the clinic) in rat serum and 0.29-2.1 g/kg (corresponding to 3.22-23.33 g in the clinic) in feces. Then, 33 potential biomarkers were identified in total. Functional pathway analysis revealed that the alterations of these biomarkers were associated with 15 metabolic pathways, mainly including arachidonic acid metabolism, glycerophospholipid metabolism, steroid biosynthesis, primary bile acid biosynthesis and sphingolipid metabolism. Of note, different doses of rhubarb could alleviate endogenous disorders to varying degrees through regulating multiple perturbed pathways to the normal state, which might be in a dose-dependent manner and involved in therapeutic mechanisms. To sum up, integrated serum and fecal metabolomics obtained that rhubarb ranging from 0.31 to 2.1 g/kg is safe and effective for constipation treatment. Also, our findings showed that the robust metabolomics techniques would be promising to be more accurately used in the dose-effect studies of complex TCM, and to clarify syndrome pathogenesis and action mechanisms in Chinese medicine.

Elucidating dosage-effect relationship of different efficacy of rhubarb in constipation model rats by factor analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhei Radix et Rhizoma (rhubarb), as the preferred representative of cathartic drugs of traditional Chinese medicine (TCM), has a long history of medicinal use and multifarious functions that produce a wide range of dosage. In modern times, rhubarb and its prescriptions are not only used to treat common clinical diseases, but also achieve good results in the treatment of acute, dangerous, severe and difficult diseases. However, rhubarb also has an alias called "General", which means that its efficacy is relatively rapid. AIM OF THE STUDY: The present study was conducted to simultaneously elucidate dosage-effect relationship of rhubarb of different efficacy, "Removing accumulation with purgation" (E1) and "Clearing heat and purging fire" (E2), providing reference for the safe and effective usage of rhubarb. MATERIALS AND METHODS: Three-week-old rats were randomly divided into the normal control group (Con.), model group (Mod.) and rhubarb groups with six doses (0.135, 0.27, 0.81, 1.35, 4.05, 8.1 g/kg). We established a constipation model with gastrointestinal accumulated heat induced by dyspepsia, taking defecation characteristics observed by metabolic cages, alvine pushing rate, gastrointestinal hormones in serum, etc., as indicators of E1, and taking TG, Na+-K+-ATPase, inflammatory factors and proteins, etc., as indicators of E2. The factor analytic approach was used to systematically evaluate the two effects and analyze the corresponding dosage-effect relationship. RESULTS: The levels of Gas, AchE, TG, Na+-K+-ATPase, TNF-α, IL-1ß, (p-)NF-κB p65, (p-)p38, (p-)ERK and p-JNK in model rats increased significantly while the levels of defecation, fecal water content, MTL, SS, ET, NTS, VIP, JNK and TLR4 decreased. Compared with the Mod., in rhubarb groups, the increase of faeces, alvine pushing rate, most gastrointestinal hormones, etc., reflected the therapeutic efficacy of E1, and the reduction of TG, Na+-K+-ATPase, expression levels of inflammatory indexes, etc., reflected the impact of E2. After the analysis, the effective threshold dose ranging from 0.67 to 5.37 g/kg (corresponding to 7.44-59.67 g in the clinic) was in the EC20-EC80 range for E1 treatment and from 0.78 to 5.60 g/kg (equivalently clinical 8.67-62.22 g) was for E2 treatment. And the 1.6- and 1.2-fold rhubarb highest dose of Chinese Pharmacopoeia might be the optimal doses for E1 and E2 respectively. In general, however, the concentration of rhubarb liquid for overall efficacy is suggested to be between middle and highest dose of Chinese Pharmacopoeia. CONCLUSION: A constipation model was used to elucidate two main effects of rhubarb, which was consistent with the characteristics of TCM syndrome. In the wide range of rhubarb dosage, low doses might have little or no effect and although high concentrations of rhubarb liquid enhanced curative efficacy, it would also have certain side effects on the body. Therefore, scientific-based experiments and rational analysis by mathematical models could contribute to the safe and effective application of rhubarb in the clinic.

Studies of the Anti-amnesic Effects and Mechanisms of Single and Combined Use of Donepezil and Ginkgo Ketoester Tablet on Scopolamine-Induced Memory Impairment in Mice.

Ginkgo ketoester tablets (GT) and donepezil were a clinically used combination for the treatment of Alzheimer's disease (AD). The aim of the study was undertaken to investigate the antiamnesic effects of the two drugs alone and in combination through in vivo models of the Morris water maze along with in vitro antioxidants, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The potential mechanisms were speculated by the activities of acetylcholine (ACh), AChE, superoxide dismutase (SOD), and malondialdehyde (MDA) and the protein expression of brain-derived neurotrophic factor (BDNF) and tyrosine protein kinase B (TrkB). The combination group showed a concentration-dependent inhibition of cholinesterase and antioxidation. As far as its mechanism was concerned, the combination of two drugs exerted excellent effects on oxidative stress, cholinergic pathway damage, and inactivation of the BDNF-TrkB signaling pathway. Additionally, to elucidate the binding mechanism of GT active ingredients into the structure of AChE, the results of molecular docking studies indicated that hydrogen and/or hydrophobic bonds might play an important role in their binding process. Thus, the combination of drugs could treat AD perfectly and further verify the scientific rationality of clinical medication.

Macrophage migration inhibitory factor promoter polymorphisms (-794CATT5-7 ) as potential biomarker for early-stage cervical cancer.

AIM: The objective was to investigate the correlation between macrophage migration inhibitory factor (MIF) promoter polymorphisms (-794CATT5-7 ) and early-stage cervical cancer (ESCC) and to identify a potential biomarker for ESCC. METHODS: A hospital-based case-control study was performed. The case group contained 250 patients with histologically confirmed ESCC. The control group included 147 healthy women. Polymerase chain reaction-single strand conformation polymorphism was used to genotype polymorphisms of MIF promoter -794CATT5-7 . Enzyme-linked immunosorbent assay was applied to detect serum concentration of MIF. RESULTS: The genotype distribution and allele frequency of MIF-794CATT in the ESCC group were significantly different from those in the control group (P < 0.05). The 7-CATT repeat carriers were significantly higher in the ESCC group than those in the control group (P < 0.05). The 7-CATT repeat carriers (5/7, 6/7, and 7/7) were associated with ESCC and increased risks of cervical cancer (odds ratio = 3.5, 3.0, and 5.6; 95% confidence interval = 1.2-10.5, 1.2-7.9, and 1.3-25.3, respectively). Serum concentration of MIF was significantly higher in the ESCC group than in the control group (P < 0.05), and it was significantly higher in 7-CATT carriers than in non-7-CATT carriers (P < 0.05). Neither polymorphisms of MIF-794 nor serum MIF were associated with lymph node metastases and differentiation (P > 0.05). CONCLUSION: MIF promoter polymorphisms (-794CATT) were correlated with ESCC; and 7-CATT might play a role in ESCC. It could be a potential biomarker for ESCC.

Correlation of polymorphism of IL-8 and MMP-7 with occurrence and lymph node metastasis of early stage cervical cancer.

The IL-8 and MMP-7 genes participate in the carcinogenesis of many malignancies, but the role of both genes in cervical cancer is not fully elucidated. The aim of this study was to determine the frequency of IL-8 and MMP-7 gene mutations and to assess their effects on the risk of early stage cervical cancer and lymph node metastasis. The clinical stage and histological grade of cervical cancer were also studied. The peripheral blood from the patients with early stage cervical cancers and normal controls was collected and the DNA was extracted. The incidence of IL-8 and MMP-7 gene mutations was assessed by using tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS PCR) and restriction fragment length polymorphism (RFLP). The data were statistically analyzed by x2 test. The results showed that: (1) The genotype frequency of IL-8 -251AT and TT was significantly higher in the cervical cancer group than in the normal control group (OR=2.290 and 2.619 respectively, P=0.001), and it was also higher in the lymphatic metastasis group than that without metastasis (OR=2.917, P=0.035); (2) The frequency of MMP-7 -181G/G genotype was significantly higher in the cervical cancer group and in the lymphatic metastasis group (P<0.05); (3) The incidence of IL-8 mutation was two times higher in IIa cervical cancer group than in Ib1 and Ib2 cervical cancer group (P=0.006). For the MMP-7 gene, there was statistically significant difference in the incidence of mutation between the Ib1, Ib2 and the IIa (P=0.000); (4) Different histological types and different grades of cervical cancer had different incidence of mutations, statistically. It was suggested that there was significant difference in the genotype of IL-8 -251TT and MMP-7 -181GG polymorphism between the cervical cancer group and the lymph node metastasis group. Moreover, individuals with IL-8 T allele or MMP-7 G allele carriers were at significantly higher risk of cervical cancer, particularly the early (IIa) and medium, poorly differentiated cervical cancer (G2+G3).

Correlation of macrophage migration inhibitory factor gene polymorphism with the risk of early-stage cervical cancer and lymphatic metastasis.

Associations of functional single nucleotide polymorphisms in MIF-173G/C with early stage cervical cancer were investigated in a hospital-based case-control study on 250 patients with cervical cancer prior to surgery (including 49 cases with and 201 cases without lymphatic metastasis) and 147 healthy controls. The polymorphism was assessed using restriction fragment length polymorphism polymerase chain reaction, and the MIF serum concentration was examined using the enzyme-linked immunosorbent assay to analyze the correlation between the polymorphism and the MIF serum concentration. Carriers of the variant C allele in MIF-173 were at a significantly higher risk of cervical cancer compared to carriers of the wild-type allele (aOR=1.508; 95% CI, 1.128-2.016, p=0.05). The GC and CC genotypes may be the causative factors for cervical cancer (aOR=1.851; 95% CI, 1.132-3.027, p=0.013). Individuals with the GC+CC genotype and C allele at the MIF-173G/C site were at a significantly higher risk of cervical cancer and lymphatic metastasis. The risk of lymphatic metastasis in early stage cervical cancer was increased more than 1.6 times in patients with the CC and GC genotypes compared with those with the GG genotype. The genotype distribution and allele frequency of MIF-173G/C were statistically significant in the well-, moderately and poorly differentiated groups (P<0.05). Compared to the GG genotype and G allele, patients with GC and CC genotypes and C allele exhibited a lower degree of differentiation and a higher degree of malignancy. A significant difference was observed in MIF serum concentrations among the various subgroups (P<0.05). The early cervical cancer, lymphatic metastasis and poorly differentiated groups exhibited higher MIF levels in serum. Moreover, patients with the CC genotype exhibited higher MIF serum concentration, which could increase the risk of early stage cervical cancer and lymphatic metastasis. The results presented in this study provide the first evidence that the genetic polymorphism MIF-173 is associated with cervical cancer in humans. Detection of MIF serum concentration and genotyping may be used as biomarkers for early diagnosis and therapy for cervical cancer.

Correlation of Polymorphism of IL-8 and MMP-7 with Occurrence and Lymph Node Metastasis of Early Stage Cervical Cancer / 华中科技大学学报（医学）（英德文版）

The IL-8 and MMP-7 genes participate in the carcinogenesis of many malignancies,but the role of both genes in cervical cancer is not fully elucidated.The aim of this study was to determine the frequency of IL-8 and MMP-7 gene mutations and to assess their effects on the risk of early stage cervical cancer and lymph node metastasis.The clinical stage and histological grade of cervical cancer were also studied.The peripheral blood from the patients with early stage cervical cancers and normal controls was collected and the DNA was extracted.The incidence of IL-8 and MMP-7 gene mutations was assessed by using tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS PCR) and restriction fragment length polymorphism (RFLP).The data were statistically analyzed by x2 test.The results showed that:(1) The genotype frequency of IL-8 -251AT and TT was significantly higher in the cervical cancer group than in the normal control group (OR=2.290 and 2.619 respectively,P=0.001),and it was also higher in the lymphatic metastasis group than that without metastasis (OR=2.917,P=0.035); (2) The frequency of MMP-7 -181G/G genotype was significantly higher in the cervical cancer group and in the lymphatic metastasis group (P＜0.05); (3) The incidence of IL-8 mutation was two times higher in Ⅱa cervical cancer group than in Ⅰ bl and Ⅰ b2 cervical cancer group (P=0.006).For the MMP-7 gene,there was statistically significant difference in the incidence of mutation between the Ⅰ bl,Ⅰ b2 and the Ⅱ a (P=0.000); (4) Different histological types and different grades of cervical cancer had different incidence of mutations,statistically.It was suggested that there was significant difference in the genotype of IL-8 -251TT and MMP-7 -181GG polymorphism between the cervical cancer group and the lymph node metastasis group.Moreover,individuals with IL-8 T allele or MMP-7 G allele carriers were at significantly higher risk of cervical cancer,particularly the early ( Ⅱ a) and medium,poorly differentiated cervical cancer (G2+G3).