Objective:To explore the effects of Yishen Tonglong Granules (YSTLG) on cell proliferation and apoptosis in a nude mouse model of androgen independent prostate cancer subcutaneous transplantation based on non classical Wnt signaling pathway.Methods:The tumor-bearing nude mouse model was established using the human prostate cancer bone metastatic cell line PC-3. After successful modeling, the mice were equally divided into six groups using the random number table method: model group, Western medicine group, Chinese materia medica low-, medium-, and high-dosage groups, and Chinese materia medica-Western medicine combination group. Corresponding drug interventions were administered to each group. Following 28 consecutive days of drug administration, the nude mice were euthanized. Tumor tissues were harvested for pathological observation via HE staining. Cell proliferation was assessed by immunohistochemistry; apoptosis was detected using TUNEL assay; the expressions of non-canonical Wnt signaling pathway-related proteins (Wnt5a, Rac1, RhoA, NFATc1) were analyzed through Western blot and immunohistochemical methods.Results:THE staining results demonstrated that YSTLG could effectively ameliorate pathological alterations in tumor tissues. Compared with the model group, the Chinese materia medica low-, medium-, and high-dosage groups, as well as the Chinese materia medica-Western medicine combination group, exhibited reduced proliferation indices ( P<0.01), elevated apoptosis indices ( P<0.01), down-regulated protein expressions of Wnt5a, Rac1, RhoA, and NFATc1 ( P<0.01), and decreased optical density values of Wnt5a, Rac1, RhoA, and NFATc1 ( P<0.01). These effects displayed a dosage-dependent trend. The Chinese materia medica-Western medicine combination group achieved the most pronounced therapeutic outcomes. Conclusions:YSTLG may exert inhibitory effects on the proliferation of androgen-independent prostate cancer cells and promote apoptosis, possibly through suppression of the non-canonical Wnt signaling pathway. Furthermore, its combination with Wnt signaling inhibitors may exhibit synergistic therapeutic effects.

Objective To investigate the clinical efficacy of Guhong Injection for the treatment of patients with coronary microvascular dysfunction(CMD)of qi stagnation and blood stasis syndrome.Methods Sixty cases of patients with CMD of qi stagnation and blood stasis syndrome who were admitted to Guangdong Provincial Hospital of Chinese Medicine from June 2021 to August 2022 were randomly divided into the control group and the trial group according to the random number table method,with 30 cases in each group.The control group was treated with conventional western medicine,and the trial group was treated with Guhong Injection on the basis of treatment for the control group.The course of treatment for the two groups covered 10 days.The changes in traditional Chinese medicine(TCM)syndrome scores,and levels of lipid indicators,serum inflammatory factors and endothelial factors in the two groups were observed before and after treatment.After treatment,the clinical efficacy of the two groups was evaluated.Results(1)During the trial,three cases in the control group and two cases in the trial group fell off and a total of 55 cases were finally included in the statistical analysis of efficacy,including 27 cases in the control group and 28 cases in the trial group.(2)After 10 days of treatment,the total effective rate of the trial group was 89.29%(25/28),and that of the control group was 40.74%(11/27),and the intergroup comparison(tested by chi-square test)showed that the therapeutic efficacy of the trial group was significantly superior to that of the control group(P<0.01).(3)After treatment,the TCM syndrome scores of patients in both groups were decreased compared with those before treatment(P<0.05),and the decrease in the trial group was significantly superior to that in the control group(P<0.01).(4)After treatment,the levels of lipid indicators triglyceride(TG),total cholesterol(TC),and low-density lipoprotein cholesterol(LDL-C)in the two groups of patients were decreased compared with those before treatment(P<0.05),and the level of high-density lipoprotein cholesterol(HDL-C)was increased compared with that before treatment(P<0.05).The intergroup comparison showed that the decrease of TG,TC,and LDL-C levels as well as the increase of HDL-C level in the trial group was significantly superior to that in the control group(P<0.05 or P<0.01).(5)After treatment,the serum levels of inflammatory factors high-sensitivity C-reactive protein(hs-CRP),interleukin 6(IL-6)and tumor necrosis factor α(TNF-α)in the two groups of patients were decreased compared with those before treatment(P<0.05),and the decrease in the trial group was significantly superior to that in the control group(P<0.01).(6)After treatment,the serum level of endothelial factor nitric oxide(NO)in the two groups of patients was increased(P<0.05)and the serum endothelin 1(ET-1)level was decreased compared with that before treatment(P<0.05),and the increase of serum NO level,as well as the decrease of serum ET-1 level in the trial group was significantly superior to that in the control group(P<0.05 or P<0.01).Conclusion Based on the conventional treatment in western medicine,the application of Guhong Injection in the treatment of patients with CMD of qi stagnation and blood stasis syndrome exerts remarkable efficacy,which can effectively alleviate the symptoms,regulate the levels of blood lipids,reduce the inflammatory response,and improve the endothelial function.

Aim To investigate the correlation between serum remnant lipoprotein cholesterol(RLP-C),triglyc-eride levels(TG)and coronary heart disease(CHD)in middle-aged people.Methods A total of 439 middle-aged in-dividuals who were hospitalized in the Department of Cardiology of Liuzhou People's Hospital from January 2015 to Decem-ber 2022 and underwent coronary angiography were selected as the research subjects.They were divided into CHD group(190 cases)and control group(249 cases)according to the results of coronary angiography.The general clinical data and laboratory tests of the subjects were collected,and RLP-C was calculated based on blood lipid profile.Bivariate Spearman correlation,multivariate Logistic regression,and restricted cubic spline graph were used to analyze the correlation between RLP-C,TG,and CHD in these middle-aged participants.Receiver operating characteristic(ROC)curve was used to evaluate the value of RLP-C and TG in predicting CHD.Results The age in CHD group was older than that in control group,proportion of male,proportion of smoking history,incidence of hypertension,incidence of diabetes,inci-dence of hyperlipidemia,body mass index(BMI),systolic blood pressure(SBP),fasting blood glucose(FBG),glycosy-lated hemoglobin(HbA1c),TG,low density lipoprotein cholesterol(LDLC),RLP-C were higher than those in control group,while high density lipoprotein cholesterol(HDLC)was lower than that in control group(P＜0.05).The Spearman correlation analysis results showed positive correlation between RLP-C,TG,LDLC and CHD(r=0.227,0.279,and 0.105,respectively,P＜0.05),and negative correlation between HDLC and CHD(r=-0.340,P＜0.001)in these stud-ied population.Multivariate Logistic regression analysis showed that whether as continuous or categorical variables,RLP-C and TG were independent risk factors for CHD(P＜0.05),HDLC was independent protective factor for CHD(P＜0.05).Compared with lowest quartile group,The OR(95％CI)of CHD incidence in 3rd and 4th quartile group of RLP-C were 2.648(1.364～5.144)and 2.847(1.468-5.520)respectively;The OR(95％CI)of CHD incidence in 3rd and 4th quartile group of TG were 3.043(1.520-6.092)and 3.520(1.811～6.842)respectively.The restricted cubic spline graph revealed that RLP-C,TG were positively nonlinearly correlated with CHD(P for overall＜0.001,P for nonlin-ear=0.002,0.001,respectively).Subgroup analysis showed that the relationship between RLP-C,TG and CHD was more significant in females than in males.ROC curve analysis showed that the areas under the curve(95％CI)of RLP-C,TG in predicting CHD were 0.632(0.580-0.685)(P＜0.001)and 0.663(0.612-0.713)(P＜0.001)in general,meanwhile,0.735(0.659-0.811)(P＜0.001)and 0.740(0.666-0.813)(P＜0.001)in females.Conclusion RLP-C and TG are independent risk factors for CHD in middle-aged people,and their correlation with CHD are greater than that of LDLC.They may become the main targets for the prevention and treatment of CHD,and should be given clinical attention.

Objective To construct a predictive model for septic shock based on the propensity score matching (PSM) method and validate its effectiveness. Methods A total of 114 patients with sepsis were enrolled as study objects, and were divided into septic shock group (40 patients) and sepsis group (74 patients) according to whether they developed septic shock. PSM was performed with a ratio of septic shock to sepsis of 1∶2, resulting in the inclusion of 30 patients in the septic shock group and 60 patients in the sepsis group after matching. The levels of C-reactive protein (CRP), procalcitonin (PCT), interleukin-6 (IL-6), serum amyloid A (SAA), soluble endothelial protein C receptor (sEPCR), endothelial cell-specific molecule 1 (ESM-1), clusterin (CLU), and the Acute Physiology and Chronic Health Evaluation Ⅱ (APACHE Ⅱ) score and Sequential Organ Failure Assessment (SOFA) score at admission were compared between the two groups. Cox proportional hazards regression analysis was used to identify the factors influencing septic shock, and a predictive model for septic shock was constructed and internally validated using the receiver operating characteristic (ROC) curve. Kaplan-Meier survival curves were plotted to analyze the differences in survival prognosis among patients with different expression levels of the indicators. Results After matching, there were no statistically significant differences in general information between the two groups (P>0.05). At admission, the septic shock group had higher levels of serum PCT, CRP, SAA, IL-6, sEPCR, ESM-1, and higher APACHE Ⅱ and SOFA scores, as well as a lower level of serum CLU compared with the sepsis group (P < 0.05). Cox regression analysis showed that PCT, CRP, SAA, IL-6, sEPCR, ESM-1, APACHE Ⅱ score, and SOFA score were independent risk factors for septic shock (P < 0.05), while CLU was an independent protective factor (P < 0.05). The predictive model for septic shock, constructed based on these factors, showed an internal validation accuracy of 94.44%, an area under the curve of 0.950, a sensitivity of 93.33%, and a specificity of 96.67%. Dead patients had higher levels of PCT, CRP, SAA, IL-6, sEPCR, ESM-1, and higher APACHE Ⅱ and SOFA scores, as well as a lower level of CLU at admission compared with survivors (P < 0.05). Compared with patients with low expression levels or low scores, patients with high expression levels of PCT, CRP, SAA, IL-6, sEPCR, ESM-1, and high APACHE Ⅱ and SOFA scores had higher fatality rates, while patients with high CLU expression levels had a lower fatality rate (P < 0.05). Conclusion The serum biomarkers including PCT, CRP, SAA, IL-6, sEPCR, ESM-1, CLU, and the APACHE Ⅱ and SOFA scores in sepsis patients are closely related to the occurrence of septic shock and survival prognosis. The predictive model constructed by combining these indicators can accurately predict the occurrence of septic shock.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, belonging to the type II CRISPR/Cas system, is an effective gene-editing tool widely used in different organisms, but the size of Streptococcus pyogenes Cas9 (SpCas9) is quite large (4.3 kb), which is not convenient for vector delivery. In this study, we used a codon-optimized Staphylococcus aureus Cas9 (SaCas9) system to edit the tyrosinase (tyr), oculocutaneous albinism II (oca2), and paired box 6.1 (pax6.1) genes in the fish model medaka(Oryzias latipes), in which the size of SaCas9 (3.3 kb) is much smaller and the necessary protospacer-adjacent motif (PAM) sequence is 5'-NNGRRT-3'. We also used a transfer RNA (tRNA)‍-single-guide RNA (sgRNA) system to express the functional sgRNA by transcription eitherin vivo or in vitro, and the combination of SaCas9 and tRNA-sgRNA was used to edit the tyr gene in the medaka genome. The SaCas9/sgRNA and SaCas9/tRNA-sgRNA systems were shown to edit the medaka genome effectively, while the PAM sequence is an essential part for the efficiency of editing. Besides, tRNA can improve the flexibility of the system by enabling the sgRNA to be controlled by a common promoter such as cytomegalovirus. Moreover, the all-in-one cassette cytomegalovirus (CMV)‍-SaCas9-tRNA-sgRNA-tRNA is functional in medaka gene editing. Taken together, the codon-optimized SaCas9 system provides an alternative and smaller tool to edit the medaka genome and potentially other fish genomes.
Animals ; Oryzias/genetics* ; Gene Editing/methods* ; CRISPR-Cas Systems ; Codon ; RNA, Guide, CRISPR-Cas Systems/genetics* ; Monophenol Monooxygenase/genetics* ; CRISPR-Associated Protein 9/genetics* ; RNA, Transfer/genetics* ; Staphylococcus aureus/genetics* ; PAX6 Transcription Factor/genetics*

【Objective】 To investigate the mechanism of Yishen Tonglong Decoction (益肾通癃汤, YSTLD) inhibiting the toll-like receptor 4/p38 mitogen activated protein kinases/nuclear factor kappa-B (TLR4/p38 MAPK/NF-κB) signaling pathway against prostate cancer by up-regulating miR-145-5p. 【Methods】 miRNA microarray technology was used to detect the changes of miRNA expression profile in prostate cancer PC-3 cells treated with YSTLD, and miRNAs with marked differences in miRNA microarray results were screened and validated by real-time polymerase chain reaction (qRT-PCR). Lentiviral transfection of miR-145-5p into prostate cancer PC-3 cells, Cell Counting Kit-8 (CCK8) assay, and scratch assay were adopted to detect the effects of miR-145-5p on prostate cancer PC-3 cell proliferation and migration. qRT-PCR and Western blot were employed to detect the effects of miR-145-5p on TLR4/p38 MAPK/NF-κB signaling pathway and the expression levels of apoptosis-related genes caspase3, tumor necrosis factor-α (TNF-α), Bax, and Bcl-2. qRT-PCR and Western blot were used to detect the effects of serum containing YSTLD on miR-145-5p, TLR4/p38 MAPK/NF-κB signaling pathway, and the expression levels of apoptosis-related genes caspase3, TNF-α, Bax, and Bcl-2. 【Results】 The expression levels of 35 miRNAs in prostate cancer PC-3 cells treated with YSTLD were significantly different from those in the control group, with miR-145-5p being the most significantly different; qRT-PCR validation revealed that the miR-145-5p levels in prostate cancer PC-3 cells treated with YSTLD were significantly higher than those in the DMSO control group (P < 0.05). After lentiviral transfection of miR-145-5p into prostate cancer PC-3 cells, miR-145-5p was found to inhibit the proliferation and migration of prostate cancer PC-3 cells. Overexpression of miR-145-5p up-regulated expression levels of caspase3, TNF-α, and Bax mRNA, and down-regulated expression levels of p38 MAPK, p65 NF-κB, and Bcl-2 mRNA in prostate cancer PC-3 cells (P < 0.05), while up-regulated caspase3 protein expression levels in prostate cancer PC-3 cells and down-regulated expression levels of TLR4, p38 MAPK, and p65 NF-κB protein (P < 0.05). Serum containing YSTLD could up-regulate the expression levels of caspase3, TNF-α, and Bax mRNA, and down-regulate the mRNA expression levels of p38 MAPK, p65 NF-κB, Bcl-2, and TNF receptor-associated factor 1 (TRAF1) in prostate cancer PC-3 cells after intervening prostate cancer PC-3 cells (P < 0.05). Simultaneously, it up-regulated the expression levels of caspase3 protein and down-regulated the protein expression levels of TLR4, p38 MARK, p65 NF-κB, and TRAF1 in prostate cancer PC-3 cells (P < 0.05). 【Conclusion】 YSTLD can promote apoptosis of prostate cancer PC-3 cells by up-regulating the expression level of miR-145-5p and inhibiting TLR4/p38 MAPK/NF-κB signaling pathway, which may be an important mechanism of YSTLD against prostate cancer.

Generation of mutants with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA (mRNA) or protein and transcribed guide RNA (gRNA). However, the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present. In this study, we employed a poly-transfer RNA (tRNA)-gRNA (PTG) system driven by cytomegalovirus (CMV) promoter to target the medaka (Oryzias latipes) endogenous gene tyrosinase(tyr) or paired box 6.1 (pax6.1) and illustrated its function in a medaka cell line and embryos. The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka. This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.
Animals ; CRISPR-Cas Systems ; Cell Line ; Gene Editing ; Oryzias/genetics* ; RNA, Guide/genetics* ; RNA, Transfer/genetics*

BACKGROUND:There are a lot of studies on isolation and culture methods of human placental chorionic-derived mesenchymal stem cells (hpcMSCs), but how to simply and efficiently harvest a large amount of primary MSCs has not been resolved. OBJECTIVE:To optimize the tissue explants method of isolating and culturing hpcMSCsin vitro. METHODS:Human placental chorionic villi were collected from full-term deliveries under aseptic condition and isolated by electric homogenizer. hpcMSCs were prepared by tissue explants method. The fluid and tissue of the primary culture flask and douching normal saline of the initial culture were centrifuged and prepared for secondary culture. RESULTS AND CONCLUSION: It saved time and effort to treat human placental chorionic villi with electric homogenizer, with good effects on tissue dispersion and removal of red blood cells. The average time of cell acquisition in initial culture and secondary culture was (17.73±1.14) and (10.03±1.30) days, respectively. The yields of primary cultured cells in initial culture and secondary culture were (6.97±0.98)×105 and (13.82±1.44)×105per Φ100 mm culture dish, respectively. The adherent cells showed fibroblast-cell-like shape, which were in parallel or circinate arrangement. Highly expressed CD73, CD105 and CD90 could be detected in the third generation of hpcMSCs, but CD34, CD45, CD14, CD19 and HLA-DR were negative. Following induction, alizarin red staining and oil red O staining produced a strong reaction in cells. In a word, the optimized method is a simple and efficient method for obtaining a large amount of primary hpcMSCs.

Objective To establish a cultivating method for obtaining a large number of P0 generation human placental chorionic-derived mesenchymal stem cells (hpcMSCs).Methods The hpcMSCs were isolated from human placental chorion.After primary culturing and culturing for seven days,the culture medium,the non-adherent tissue and the douching normal saline of the primary culture were centrifuged and re-cultured twice.Cell morphology was observed by an inverted microscope.CCK-8 was used to measure the cell growth curve.Flow cytometry was used to detect cell surface markers.Adipogenic and osteogenic differentiation kits were used to assess the cell differentiation potential.Results The obtained hpcMSCs were fibroblast-like adherent cells and (25.54±3.38)×106 cells were obtained per placenta.The total yield of the primary culture,secondary culture and tertiary culture were (11.73±2.09)×106,(11.12±1.42)×106 and (2.69±0.71)×106,respectively,and the incubation time were (12.00±0.64) d,(8.87±0.63) d and (12.33±0.80) d.There was significant differences in incubation time between the secondary culture and the primary culture as well as the tertiary culture (all P＜0.05),and there was no significant difference between the primary culture and the tertiary culture.However,the incubation time of the tertiary culture had an increasing trend (P＞0.05).The yield per culture flask of the primary culture,secondary culture and tertiary culture were (1.12±0.15) × 106,(2.10±0.16)×106 and (1.04±0.16)×106,respectively.There was significant differences in the yield per culture flask between the secondary culture and the primary culture as well as the tertiary culture (all P＜0.05),and there was no significant difference between the primary culture and the tertiary culture.However,the yield per culture flask of the tertiary culture had a decreasing trend (P＞0.05).There was no difference among the three cultures in the growth curve and the expression of surface markers,and the osteogenic and adipogenic differentiation were all positive.Conclusions The P0 generation hpcMSCs isolated from a choriocarcinoma sample can be doubled by the three cultures compared with the primary culture,which can provide plenty stem cell source for the regenerative medicine.

Aim To observe the effect of pyrimidine dithiocarbamate (PDTC) on the variance of disc morphology and the expressions of TNF-α, MMP-9 in the cervical disc in cervical dynamic equilibrium rat models, and to investigate the roles of PDTC in the process of intervertebral disc degeneration and the mechanism involved.Methods Fifty-four SD rats were divided into three groups randomly, then the dynamic equilibrium rat model was established by cutting the nuchal superficial and deep muscle of the rats.The dynamic equilibrium rats with PDTC solution intraperitoneal injection after operations were defined as PDTC group (group A), the models with saline intraperitoneal injection after operations as saline group (group B), the rats of fake operation with saline intraperitoneal injection as blank group (group C), and the animals were sacrificed in batches 10 weeks, 12 weeks, 16 weeks respectively after operation.The C5, C6 vertebrae and C5/6 discs were harvested, and the disc morphology was observed.TNF-α, MMP-9 mRNA expressions were detected by q-PCR and protein expression was observed by Western blot.Results Compared with the saline group, the morphology of disc in PDTC group was destructed slightly and fiber ring arranged orderly.TNF-α, MMP-9 gene and protein expressions had no obvious changes (P>0.05) in blank group (group C) at each time point.The expressions of IL-6, MMP-9 mRNA increased with time in group B, but the amount increased fast firstly, and slow lately, reaching peak in 12 weeks.The expression of TNF-α, MMP-9 protein became steady in group B from 10 weeks compared with other time points(P>0.05).TNF-α, MMP-9 genes and proteins expression decreased obviously in PDTC group (group A) compared with saline group (group B) (P<0.01) at each time point, but higher than blank group C(P<0.01) at each time point.Conclusions TNF-α and MMP-9 are important inflammatory factors involved in rat cervical disc degeneration, PDTC relieves the degeneration of rat cervical disc by reducing the expression of TNF-α and MMP-9 via disturbing the NF-κB signal pathway probably, and PDTC may become potential medicine for disc degeneration.