ObjectiveTo investigate the effects of different fermentation methods and times on the fungal flora and chemical composition of Aurantii Fructus， in order to obtain the optimal fermentation conditions and flora structure， and to ensure the stability and controllability of the fermented varieties. MethodsScanning electron microscopy was used to observe and analyze the colony characteristics on the surface of Aurantii Fructus under different fermentation conditions. Internal transcribed spacer 2（ITS2） high-throughput sequencing， combined with fungal community diversity analysis and fungal community structure analysis， were used to obtain the fungal flora microbial categories of Aurantii Fructus under the conditions of traditional pressure-shelf fermentation and non-pressure-shelf natural fermentation for 7， 14， 21 d（numbered Y1-Y3 for the former， and numbered F1-F3 for the latter）， respectively. At the same time， the chemical components in the fermentation process were detected by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）， combined with principal component analysis（PCA）， partial least squares-discriminant analysis（PLS-DA） and compound retention time， parent ions， characteristic fragment ions and other information， the differential compounds between the different fermentation samples were screened and identified. ResultsThe analysis of fungal community diversity showed that the dominant flora did not change at different fermentation time points in the traditional pressure-shelf fermentation method， while in the non-pressure-shelf natural fermentation method， there was a significant difference with the fermentation process， and at the genus level， the dominant genus of samples Y1， Y2， Y3 and F2 was Aspergillus， while the dominant genera of samples F1 and F3 were both Rhizopus. This indicated that the microbial growth environment provided by the traditional fermentation method was more stable， and the microbial community structure was more stable， which was more conducive to the stable and controllable fermentation process and fermented products. A total of 155 compounds were identified by compositional analysis， including 70 flavonoids， 38 coumarins， 10 alkaloids， 34 organic acids and 3 other compounds. After fermentation， two new components of ribalinine and pranferin were produced. Different fermentation conditions also brought about differences in chemical composition， multivariate statistical analysis obtained 26 differential compounds under two different fermentation methods， mainly including flavonoids， organic acids and coumarins. Comprehensively， the microbial community structure of samples fermented by the traditional pressure-shelf method of Aurantii Fructus for 14 d was stable， the species richness was high and the overall content of differential compounds was high， which was the optimal processing condition. ConclusionCompared with non-pressure-shelf natural fermentation， the traditional method has obvious advantages in terms of the stability of the microbial community structure and the content of chemical compounds， and the optimal condition is 14 days of fermentation. This study is helpful to promote the quality stability and fermentation bioavailability of fermented products of Aurantii Fructus， as well as to provide an experimental basis for the further improvement of the quality control methods of this variety.

ObjectiveTo investigate the effects of different fermentation methods and times on the fungal flora and chemical composition of Aurantii Fructus， in order to obtain the optimal fermentation conditions and flora structure， and to ensure the stability and controllability of the fermented varieties. MethodsScanning electron microscopy was used to observe and analyze the colony characteristics on the surface of Aurantii Fructus under different fermentation conditions. Internal transcribed spacer 2（ITS2） high-throughput sequencing， combined with fungal community diversity analysis and fungal community structure analysis， were used to obtain the fungal flora microbial categories of Aurantii Fructus under the conditions of traditional pressure-shelf fermentation and non-pressure-shelf natural fermentation for 7， 14， 21 d（numbered Y1-Y3 for the former， and numbered F1-F3 for the latter）， respectively. At the same time， the chemical components in the fermentation process were detected by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）， combined with principal component analysis（PCA）， partial least squares-discriminant analysis（PLS-DA） and compound retention time， parent ions， characteristic fragment ions and other information， the differential compounds between the different fermentation samples were screened and identified. ResultsThe analysis of fungal community diversity showed that the dominant flora did not change at different fermentation time points in the traditional pressure-shelf fermentation method， while in the non-pressure-shelf natural fermentation method， there was a significant difference with the fermentation process， and at the genus level， the dominant genus of samples Y1， Y2， Y3 and F2 was Aspergillus， while the dominant genera of samples F1 and F3 were both Rhizopus. This indicated that the microbial growth environment provided by the traditional fermentation method was more stable， and the microbial community structure was more stable， which was more conducive to the stable and controllable fermentation process and fermented products. A total of 155 compounds were identified by compositional analysis， including 70 flavonoids， 38 coumarins， 10 alkaloids， 34 organic acids and 3 other compounds. After fermentation， two new components of ribalinine and pranferin were produced. Different fermentation conditions also brought about differences in chemical composition， multivariate statistical analysis obtained 26 differential compounds under two different fermentation methods， mainly including flavonoids， organic acids and coumarins. Comprehensively， the microbial community structure of samples fermented by the traditional pressure-shelf method of Aurantii Fructus for 14 d was stable， the species richness was high and the overall content of differential compounds was high， which was the optimal processing condition. ConclusionCompared with non-pressure-shelf natural fermentation， the traditional method has obvious advantages in terms of the stability of the microbial community structure and the content of chemical compounds， and the optimal condition is 14 days of fermentation. This study is helpful to promote the quality stability and fermentation bioavailability of fermented products of Aurantii Fructus， as well as to provide an experimental basis for the further improvement of the quality control methods of this variety.

Pyroptosis is the programmed death of cells accompanied by an inflammatory response and is widely involved in the development of a variety of diseases, such as infectious diseases, cardiovascular diseases, and neurodegeneration. It has been shown that cellular scorching is involved in the pathogenesis of pulmonary arterial hypertension ( PAH) in cardiovascular diseases. Patients with PAH have perivascular inflammatory infiltrates in lungs, pulmonary vasculopathy exists in an extremely inflam-matory microenvironment, and pro-inflammatory factors in cellular scorching drive pulmonary vascular remodelling in PAH patients. This article reviews the role of cellular scorch in the pathogenesis of PAH and the related research on drugs for the treatment of PAH, with the aim of providing new ideas for clinical treatment of PAH.

Tropane alkaloids (TAs) are a class of anticholinergic drugs widely used in clinical practice and mainly extracted from plant, among which Atopa belladonna is the main commercial drug source. It is of great industrial value to obtain TAs in large quantities by plant metabolic engineering. In TAs pathway, cytochrome oxidase CYP82M3 catalyze the synthesis of tropinone and then tropinone reductase I (TRI) compete with TRII for tropinone to form tropine leading to the TAs synthesis (drainage). In this study, based on the "increasing flow and drainage" metabolic engineering strategy, two genes, namely HnCYP82M3 and DsTRI from Hyoscyamus niger and Datura stramonium, respectively, were overexpressed in the hair roots of A. belladonna, with a view to promote the TAs accumulation. The HnCYP82M3 gene was cloned from the root of H. niger, and it encoded amino acid with 91.7% sequence identity with AbCYP82M3 from A. belladonna. Overexpression of HnCYP82M3 alone did not affect the content of TAs in hair roots of A. belladonna, indicating that CYP82M3 was not a key enzyme in TAs biosynthesis. Simultaneous overexpression of HnCYP82M3 and DsTRI greatly promoted the accumulation of the three TAs, and the contents of hyoscyamine, anisodamine and scopolamine were 4.97 times, 2.83 times and 2.19 times that of the control, respectively, and the increase amplitude was greater than that of single overexpression of DsTRI. This study showed that the "increasing flow and drainage" strategy of enzyme genes co-expression at branch points was a promising metabolic engineering method to effectively improve the biosynthesis of TAs in A. belladonna, and laid a theoretical and technical foundation for the large-scale industrial acquisition of TAs.

Objective To investigate the anti-fibrosis action of the therapy of tonifying kidney and removing stasis(shortened to Bushen Quyu method)on moderate-to-severe intrauterine adhesion and its influence on extracellular matrix degradation related factors in menstrual blood.Methods A total of 124 patients with moderate-to-severe intrauterine adhesions of kidney deficiency and blood stasis syndrome who were to undergo transcervical resection of adhesion(TCRA)were randomly divided into a study group and a control group,with 62 patients in each group.Both groups were given TCRA treatment.After the operation,the control group was treated with Progesterone + Estradiol Valerate Tablets,and the study group was treated with the combination of the Bushen Quyu method by using modified Guishen Decoction based on the treatment of the control group.The treatment covered 3 menstrual cycles.The changes in menstrual status,transvaginal color Doppler sonography parameters,scores of kidney deficiency and blood stasis syndrome,and scores of uterine adhesion in the two groups were observed before and after the treatment.Moreover,the two groups were observed before and after treatment in the changes of vascular endothelial growth factor(VEGF),transforming growth factor β1(TGF-β1),platelet-derived growth factor(PDGF),and connective tissue growth factor(CTGF)as well as the mRNA expression levels of matrix metalloproteinase 9(MMP-9),matrix metalloproteinase inhibitory factor 1(TIMP-1),phosphatidylinositol-3-hydroxykinase(PI3K)and collagen type I A1 protein(COL1A1).After treatment,the clinical efficacy and safety of the two groups were assessed.Results(1)After 3 menstrual cycles of treatment,the total effective rate in the study group was 93.55%(58/62),and that in the control group was 80.65%(50/62).The intergroup comparison showed that the therapeutic effect of the study group was significantly superior to that of the control group(P＜0.05).(2)After treatment,the menstrual flow,menstrual cycle and menstrual period of the two groups were significantly improved compared with those before treatment(P＜0.05),and the recovery of menstrual flow,menstrual cycle and menstrual period in the study group was significantly superior to that in the control group after treatment(P＜0.05).(3)After treatment,the transvaginal color Doppler sonography parameters of uterine blood flow index(FI),pulsatility index(PI),resistance index(RI),uterine cavity volume,and endometrial thickness in the two groups were significantly improved compared with those before treatment(P＜0.05).And the study group had significantly higher FI,uterine cavity volume,and endometrial thickness and had lower PI and RI than the control group(P＜0.05 or P＜0.01).(4)After treatment,the scores of uterine adhesion and the scores of kidney deficiency and blood stasis syndrome in the two groups were significantly decreased compared with those before treatment(P＜0.05),and the above scores in the study group were significantly lower than those of the control group after treatment(P＜0.01).(5)After treatment,the levels of VEGF,TGF-β1,PDGF,and CTGF in the two groups were significantly decreased compared with those before treatment(P＜0.05),and the levels of the above cytokines in the study group were significantly lower than those in the control group after treatment(P＜0.01).(6)After treatment,the relative mRNA expression levels of MMP-9 and PI3K in the menstrual blood of the two groups were significantly higher and the mRNA expression level of COL1A1 was significantly lower than that before treatment(P＜0.05).And the study group had higher mRNA expression levels of MMP-9 and PI3K in the menstrual blood than the control group after treatment(P＜0.05 or P＜0.01).(7)The total incidence of adverse reactions in the control group was 25.81%(16/62)and that in the study group was 16.13%(10/62).The intergroup comparison showed that the difference between the two groups was not statistically significant(P＞0.05).The symptoms of adverse reactions in the two groups disappeared after symptomatic treatment,and there were no adverse reactions related to the medication of Chinese medicine during the study.Conclusion The combination of Bushen Quyu formula combined with western medicine is more effective than western medicine alone for the treatment of patients with uterine adhesions after TCRA.The application of Bushen Quyu formula can further promote the recovery of menstruation in the patients,improve the circulation of blood flow in the endometrium,increase the endometrial thickness,regulate the expression of fibrosis factors,up-regulate the mRNA expressions of PI3K and MMP-9,restore the balance of the extracellular matrix(ECM),and prevent the reoccurrence of intrauterine adhesions.And its clinical medication is safe.

BACKGROUND:Deferoxamine mesylate is a potential anti-osteoporosis drug with iron chelation,vascular regeneration,and antioxidant effects.Recent studies have shown that the application of deferoxamine mesylate can be extended to the field of tissue regeneration engineering. OBJECTIVE:To investigate whether deferoxamine mesylate can promote the repair effect of iron overload osteoporotic rats after bone grafting for mandibular bone defects by simulating the state of iron accumulation in patients with postmenopausal osteoporosis with high iron intervention in osteoporotic rats. METHODS:An iron accumulation ovariectomized osteoporosis model was firstly constructed.The model group underwent bilateral ovariectomy,and the intraperitoneal injection of ferric ammonium citrate(90 mg/kg,twice a week,for 11 weeks)was started in the 2nd week,while the sham-operated group had some fat around the ovaries removed and was given an equal amount of saline for 11 weeks.After the successful modeling,the experimental rats were divided into sham-operated group(n=6),high iron ovariectomtized group(n=6)and high iron ovariectomized deferoxamine mesylate treatment group(deferoxamine mesylate group,n=6).Bone defects of 5 mm in diameter were established in the rat's bilateral mandibles and implanted with Bio-Oss bone powder.Intraperitoneal injection of deferoxamine mesylate(100 mg/kg,3 times a week)was started on postoperative day 4 in the deferoxamine mesylate group,and equal volume of saline was given in the sham-operated and high iron ovariectomized groups.The bone samples of the mandible,liver and blood were taken at 2 and 12 weeks after bone grafting for Prussian blue staining of the jaw and liver and ELISA detection of serum ferritin to detect iron levels in various body tissues;hematoxylin-eosin staining and Masson staining were performed to observe inflammatory cell infiltration and early osteogenesis in the bone defect area;tartrate resistant acid phosphatase staining was performed to observe osteoclast differentiation;ELISA was performed to detect serum calcitonin and type I collagen C-terminal peptide levels;and Micro-CT and hematoxylin-eosin staining were performed to observe osteogenesis in the middle and late stages. RESULTS AND CONCLUSION:The number of tibial trabeculae was reduced and the trabeculae were sparsely arranged in the high iron ovariectomized group.Iron levels in the liver,jaw bone and serum were significantly higher in the high iron ovariectomized group than the sham-operated group at 2 weeks after bone grafting,while the iron levels were significantly decreased after deferoxamine mesylate intervention(P＜0.05).In the early stage of bone defect repair,more inflammatory cell infiltration,less new bone matrix and less type I collagen fiber production were observed in the high iron ovariectomized group than in the sham-operated group(P＜0.05);after deferoxamine mesylate treatment,inflammatory cell infiltration was reduced,a small amount of new bone matrix was produced and collagen fibers increased significantly(P＜0.05).In the middle and late stages of bone defect repair,Micro-CT results showed a reduction in new bone production in the high iron ovariectomized group compared with the sham-operated group and increased new bone matrix after deferoxamine mesylate treatment(P＜0.05).Compared with the sham-operated group,the osteoclast number,serum calcitonin level,and serum type I collagen C-terminal peptide level were increased in the high-iron ovariectomized group,while the osteoclast number was decreased and bone metabolic indexes were improved after treatment with deferoxamine mesylate.To conclude,in ovariectomized rats with high iron intervention,elevated iron levels can be seen in multiple tissues,accompanied by reduced new bone production in the mandibular bone defect area.Deferoxamine mesylate can improve bone metabolism and inhibit osteoclast activity by removing iron deposits in tissues,improve bone formation in iron-accumulated osteoporotic rats,and promote bone healing in the mandibular bone defect area.

BACKGROUND:Interleukin-4 can promote the osteogenic effect of bone substitute materials,but its molecular mechanism is not yet clear.Further elucidating the mechanism of interleukin-4 promoting osteogenic effect can help find safe,economical,and effective methods for the regeneration treatment of alveolar bone defects in patients. OBJECTIVE:To explore the effect of interleukin-4 intervention on polarization transformation of macrophages and osteogenic differentiation of bone marrow mesenchymal stem cells and its possible mechanism. METHODS:RAW264.7 cells in the M1 group were induced with interferon gamma + lipopolysaccharide for 24 hours.RAW264.7 cells in the interleukin-4+M1 group were induced with interferon gamma + lipopolysaccharide for 24 hours and then interleukin-4 was added for 24 hours.RAW264.7 cells in the interleukin-4+AG+M1 group were induced with interferon gamma + lipopolysaccharide for 24 hours,and then interleukin-4 and AG-490,a JAK/STAT pathway inhibitor,were added for 24 hours.After intervention,immunofluorescence staining was used to analyze the expression of inducible nitric oxide synthase and CD206,the phenotypic marker protein of macrophages.ELISA kit was used to detect the expression of interleukin-10 and tumor necrosis factor-α in the supernatant of cell culture.The gene expressions of nodular receptor protein-3(NLRP3),interleukin-1β,and caspase-1 were detected by RT-qPCR.The expression levels of tyrosine protein kinase 1(JAK1)/phosphorylated tyrosine protein kinase 1(p-JAK1),signal transduction and transcription activator 6(STAT6)/phosphorylated signal transduction and transcription activator 6(p-STAT6),NLRP3,pro-interleukin-1β and pro-caspase-1 were detected by western blot assay.Then,RAW264.7 cells in the above four groups were indirectly co-cultured with bone marrow mesenchymal stem cells by transwell for 24 hours,followed by alkaline phosphatase staining and alizarin red staining.The mRNA expressions of alkaline phosphatase,collagen type I,and osteocalcin were detected by RT-qPCR. RESULTS AND CONCLUSION:(1)Immunofluorescence and ELISA results showed that interleukin-4 intervention could promote the expression of CD206 and interleukin-10 in M2 macrophages,and inhibit the secretion of inducible nitric oxide synthase and tumor necrosis factor-α.(2)RT-qPCR results showed that interleukin-4 could suppress the expression of NLRP3,interleukin-1β,and caspase-1 mRNAs.(3)Western blot assay showed that interleukin-4 could promote the expression of JAK1/p-JAK1,STAT6/p-STAT6 and NLRP3 proteins.(4)The alkaline phosphatase staining and alizarin red staining of bone marrow mesenchymal stem cells co-cultured with the interleukin-4+M1 group were significantly enhanced,and the mRNA expressions of alkaline phosphatase,collagen type I,and osteocalcin were significantly increased.It is concluded that interleukin-4 may inhibit the activation of NLRP3 by up-regulating JAK1/STAT6 pathway,thus promoting the transformation of macrophages from M1 polarization to M2 polarization,and finally enhancing the osteogenic differentiation ability of bone marrow mesenchymal stem cells.

Objective To observe the clinical characteristics of patients with moderate hyponatremia in general surgery and analyze the efficacy.Methods Patients with moderate hyponatremia in general surgery were divided into control group(without supplemented concentrated sodium chloride injection)and treatment group(supplemented concentrated sodium chloride injection)according to the cohort method.The clinical characteristics of patients with moderate hyponatremia,such as gender distribution,age distribution,nutritional status,and primary disease were analyzed.Propensity score matching(PSM)was performed to balance confounding factors between the two groups,making their baseline conditions comparable and comparing the differences in clinical outcomes between the two groups.Results A total of 227 cases were included,including 82 in the control group and 145 in the treatment group.The predisposing factors of moderate hyponatremia in general surgery patients include male,elderly,malnutrition,parenteral nutrition,and malignant tumors.In addition,after PSM,there were 73 patients in each group of the control group and treatment group with moderate hyponatremia.The total effective rate of the control group was 80.82％,and correction of the hyponatremia takes time was(3.97±2.54)The total effective rate of the treatment group was 98.63％,and correction of the hyponatremia takes time was(3.54±1.90)d.The differences were statistically significant(all P＜0.05).Conclusion We should pay more attention to patients with moderate hyponatremia in general surgery,and be alert to the risk of severe hyponatremia.For patients with moderate hyponatremia,concentrated sodium chloride injection should be given to improve efficacy and prognosis.

Objective To explore the regulatory mechanism of Macelignan on oxidative stress-mediated neuronal injury in autophagy and apoptosis.Methods Murine hippocampal neuronal HT22 cells were treated with 2.5 mmol·L-1 glutamic acid(Glu)to establish an oxidative stress cell model.The cells were divided into normal group(normal cultured cells),model group(2.5 mmol·L-1 Glu)and experimental-L,-M,-H groups(2.5,5,10 μmol·L-1Macelignan treatment),inhibitor group(2.5 mmol·L-1 Glu+10 μmol·L-1 Macelignan+10 μmol·L-1 LY294002).Aoptosis rate was detected by flow cytometry;the protein expression level of autophagy-related protein LC3B(LC3B),anti-SQSTM1/p62(p62),p21,B-cell lymphoma-2(Bcl-2)and Bcl-2 associated X protein(Bax)was detected by Western blot.Results The apoptosis rates in the normal group,model group and experimental-L,-M,-H groups were(4.58±1.25)％,(8.75±0.55)％,(6.30±1.71)％,(5.97±2.27)％and(5.49±1.71)％.The difference between model group and normal group was statistically significant(P＜0.01).The difference between experimental-L,-M,-H groups and model group was statistically significant(all P＜0.01).The levels of LC3B in normal group,model group,experimental-L,experimental-M,experimental-H groups and inhibitor group were 0.28±0.02,0.74±0.02,1.02±0.04,0.70±0.03,0.26±0.02 and 0.21±0.01;p62 levels were 0.49±0.08,0.33±0.03,0.50±0.07,0.59±0.01,0.64±0.13 and 0.65±0.06;p21 levels were 0.87±0.02,1.18±0.03,0.98±0.03,0.88±0.03,0.72±0.06 and 0.81±0.02;Bcl-2/Bax levels were 1.74±0.23,1.11±0.10,1.38±0.05,1.66±0.26,1.58±0.29 and 1.53±0.09,respectively.The differences between model group and normal group,between model group and experimental-H group,between model group and inhibitor group,were also statistically significant(all P＜0.01).Conclusion Macelignan can reduce the damage of hippocampal neurons induced by glutamate acid by regulating the process of autophagy and apoptosis,and has obvious neuroprotective effect.

Objective To investigate the effect of Ganoderma lucidum polysaccharide peptide(GLPP)on proliferation,migration and apoptosis of diffuse large B cell lymphoma(DLBCL)cells and its mechanism.Methods OCI-LY19 cells were divided into six groups:control,GLPP,si-NC,si-protein arginine methyltransferase 6(PRMT6),GLPP+pcDNA3.1-NC and GLPP+pcDNA3.1-PRMT6 groups.The si-NC,si-PRMT6,GLPP+pcDNA3.1-NC and GLPP+pcDNA3.1-PRMT6 groups were transfected with si-NC,si-PRMT6,pcDNA3.1-NC and pcDNA3.1-PRMT6,respectively.After the transfection was completed,control,si-NC and si-PRMT6 groups were treated with RPMI-1640 medium,while the GLPP,GLPP+pcDNA3.1-NC and GLPP+pcDNA3.1-PRMT6 groups were cultured with RPMI-1640 medium containing with 20 μg·mL-1 GLPP.After administration 24 h,the cell proliferation inhibition rates,mobility rates and apoptosis rates were detected.The expression levels of PRMT6 protein were measured by Western blotting.Results The cell proliferation inhibition rates of si-NC,si-PRMT6,GLPP+pcDNA3.1-NC and GLPP+pcDNA3.1-PRMT6 groups were(1.28±0.16)％,(38.61±3.29)％,(52.84±7.74)％and(22.75±3.87)％,respectively.The number of cell migrations in the control,GLPP,si-NC,si-PRMT6,GLPP+pcDNA3.1-NC and GLPP+pcDNA3.1-PRMT6 groups was(252.65±24.65),(136.54±16.46),(231.65±21.24),(142.76±15.34),(140.23±9.84)and(192.38±23.38)cells;the apoptosis rates were(4.36±0.52)％,(28.24±2.36)％,(4.23±0.45)％,(24.54±2.27)％,(28.42±3.85)％and(14.25±2.13)％);the expression levels of PRMT6 protein were 1.82±0.21,0.56±0.05,1.78±0.19,0.54±0.05,0.29±0.02 and 0.32±0.03,respectively.The differences of above indexes were statistically significant between control group and GLPP group,between si-NC group and si-PRMT6 group,between GLPP+pcDNA3.1-NC group and GLPP+pcDNA3.1-PRMT6 group(all P＜0.05).Conclusion GLPP could inhibit proliferation,migration and promote apoptosis of DLBCL cells by down-regulating PRMT6 expression.