BACKGROUND:Croton cream can activate ERK pathways and have anti-apoptotic effects on neuronal cells.It is not clear whether it synergistically exerts anti-apoptotic effects by inhibiting the activation of JNK and p38 pathways. OBJECTIVE:To explore the effects and mechanisms of croton cream on neuronal damage and apoptosis in the ischemic cortex of rats with cerebral ischemia-reperfusion injury. METHODS:(1)Ninety Sprague-Dawley rats were randomly divided into sham operation group,model group,croton cream low-dose group,croton cream medium-dose group,croton cream high-dose group and nimodipine group,with 15 rats in each group.Except for the sham operation group,animal models of middle cerebral artery occlusion were prepared in rats by the thread method.Rats in the three croton cream groups were given 20,40,and 60 mg/kg croton cream,respectively.Rats in the sham operation and model groups were given the same amount of normal saline,once a day,for 7 consecutive days.The optimal concentration of croton cream,namely the high dose of croton cream,was selected based on neurological deficit score,TTC staining,brain tissue water content,hematoxylin-eosin staining and Nissl staining.(2)Another 120 Sprague-Dawley rats were randomly divided into sham operation group,model group,croton cream group,JNK inhibitor group,croton cream+JNK inhibitor group,p38 MAPK inhibitor group,croton cream+p38 MAPK inhibitor group,and nimodipine group,with 15 rats in each group.Animal models of middle cerebral artery occlusion were prepared using the thread method in all the groups except in the sham operation group.Thirty minutes before modeling,10 μL of SP600125(JNK inhibitor)and 10 μL of SB203580(p38 MAPK inhibitor)were injected into the lateral ventricle of the rats,respectively.Rats in croton cream groups were intragastrically given 60 mg/kg croton cream.Seven days later,the JNK/p38 MAPK signaling pathway,apoptosis-related proteins and cell apoptosis were detected by western blot,TUNEL staining and flow cytometry,respectively. RESULTS AND CONCLUSION:(1)Compared with the sham operation group,neurological deficit score,cerebral water content,cerebral infarction volume and apoptosis rate were significantly increased in the model group(P<0.05),where nerve cells showed scattered distribution.Compared with the model group,neurological deficit score,water content of brain tissue and cerebral infarction volume were significantly decreased in the croton cream medium-dose group,high-dose group and nimodipine group(P<0.05),and the pathological morphology of nerve cells was significantly improved.(2)Compared with the JNK inhibitor group,p-JNK/JNK,p-p38/p38 and Bax expressions in rat brain tissue and the apoptotic rate were significantly decreased in the croton cream+inhibitor groups(P<0.05),while the expression of and Bcl-2 was significantly increased(P<0.05).To conclude,croton cream may inhibit the activation of JNK/p38 MAPK signaling pathway and reduce neuronal apoptosis to achieve neuroprotective effects in rats with cerebral ischemia-reperfusion injury.

Pulmonary hypertension (PH) is an extremely malignant pulmonary vascular disease of unknown etiology. ADAR1 is an RNA editing enzyme that converts adenosine in RNA to inosine, thereby affecting RNA expression. However, the role of ADAR1 in PH development remains unclear. In the present study, we investigated the biological role and molecular mechanism of ADAR1 in PH pulmonary vascular remodeling. Overexpression of ADAR1 aggravated PH progression and promoted the proliferation of pulmonary artery smooth muscle cells (PASMCs). Conversely, inhibition of ADAR1 produced opposite effects. High-throughput whole transcriptome sequencing showed that ADAR1 was an important regulator of circRNAs in PH. CircCDK17 level was significantly lowered in the serum of PH patients. The effects of ADAR1 on cell cycle progression and proliferation were mediated by circCDK17. ADAR1 affects the stability of circCDK17 by mediating A-to-I modification at the A5 and A293 sites of circCDK17 to prevent it from m1A modification. We demonstrate for the first time that ADAR1 contributes to the PH development, at least partially, through m1A modification of circCDK17 and the subsequent PASMCs proliferation. Our study provides a novel therapeutic strategy for treatment of PH and the evidence for circCDK17 as a potential novel marker for the diagnosis of this disease.

Objective:To prepare a fluorescent probe Cetuximab-IRDye800CW targeting epidermal growth factor receptor (EGFR) and investigate its application value in surgical navigation of glioblastoma (GBM).Methods:The fluorescence properties of Cetuximab-IRDye800CW were determined by fluorescence spectrophotometer. The specificity of Cetuximab-IRDye800CW bound to GBM cells was verified by Western blot. The competitive binding method of enzyme-linked immunosorbent assay (ELISA) was used to prove whether the probe could achieve tumor targeting by binding to EGFR. Subcutaneous models of 6 nude mice of GBM were divided into experimental group ( n=3; injected with Cetuximab-IRDye800CW) and control group ( n=3; injected with IRDye800CW), and images were obtained at 5 min, 24 h, 48 h and 72 h after injection. Differences of mean fluorescence intensity (MFI) and tumor to background ratio (TBR) between experimental group and control group were compared. In situ models of GBM nude mice were established ( n=6), and MRI and intraoperative navigation were conducted, which were compared with pathological distribution. Independent-sample t test was used to analyze the data. Results:The maximum emission wavelength of Cetuximab-IRDye800CW was 820 nm, which could be received by near infrared fluorescence imaging equipment. Western blot showed that Cetuximab-IRDye800CW was only bound to GBM cells. The competitive binding of ELISA showed that Cetuximab-IRdye800CW could achieve tumor targeting by binding with EGFR. At 5 min, 24 h, 48 h and 72 h after injection of fluorescent materials, the MFI values of experimental group were 109.00±3.81, 73.36±9.93, 55.24±8.82, 37.71±6.11, which were higher than those of control group (91.32±4.17, 42.91±5.39, 25.08±6.05, 8.33±1.00; t values: 4.36-9.40, P values: 0.011-0.049). The TBR of experimental group was higher than that of control group at 24 h and 48 h after injection (24 h: 2.40±0.28 vs 1.57±0.07, t=4.94, P=0.039; 48 h: 2.07±0.12 vs 1.22±0.08, t=9.85, P=0.010). GBM in situ model was successfully constructed and verified by MRI, and the tumor was visualized under the fluorescence device navigation. Pathological distribution of the tumor with HE staining was consistent with fluorescence imaging. Conclusion:Cetuximab-IRDye800CW has fluorescence imaging capability and can identify tumor boundaries in intraoperative navigation of GBM, which has potential clinical application value.

Objective:To explore the effective method for treatment of small and short penis after hypospadias surgery.Method:s From November 2017 to November 2018, 57 children aged 4 to 14[mean age(7.91±2.89)years] with hypospadias who met the diagnostic criteria of small penis were reexamined at the First Affiliated Hospital of Xin-xiang Medical University.They were randomly divided into the physical treatment group and the drug treatment group according to the order of visits, and the untreated patients were included in the control group.Among them, 21 patients in the physical treatment group were treated with penile rehabilitation therapy apparatus, supplemented by Salvia mil-tiorrhiza bath (30 minutes/time, once/day, 10 days), and 20 patients in the drug treatment group were treated with Testosterone cream topically (3 times/day, 10 days). Penile relaxation length, stretch length, transverse and longitudinal diameters of glans in 2 groups before and after the treatment were measured.The relevant indexes of 16 patients in the control group measured before and after 10 days and compared with those in the treatment group.Result:s (1)The penile relaxation length in the physical treatment group increased from (25.48±6.13) mm to (30.72±6.49) mm, the length of stretch increased from (34.90±7.71) mm to (41.08±8.43) mm, the transverse diameter of glans increased from (14.81±3.40) mm to (16.57±3.42) mm, and the longitudinal diameter increased from (13.94±3.15) mm to (15.82±3.52) mm, and the differences were statistically significant (all P<0.05). (2)The penile relaxation length in the drug treatment group increased from (21.07±4.26) mm to (31.32±4.72) mm, the length of stretch increased from (31.94±7.96) mm to (45.39±7.24) mm, the transverse diameter of glans increased from (13.38±1.77) mm to (16.64±2.10) mm, and the longitudinal diameter increased from (13.09±1.77) mm to (16.62±1.86) mm, and the differences were statistically significant (all P<0.05). (3)There was no significant difference in penile relaxation length, the length of stretch, transverse diameter and longitudinal diameters of glans before and after 10 days in the control group (all P>0.05). (4) Compared with the control group, the penile relaxation length, the length of stretch, transverse diameter and longitudinal diameters of glans in the physical treatment group increased significantly, and the differences of growth values between the two groups were statistically significant (all P<0.05). (5) Compared with the control group, the penile relaxation length, the length of stretch, transverse diameter and longitudinal diameters of glans in the drug treatment group also increased significantly, and the difference of growth values between the two groups were statistically significant (all P<0.05). (6) The growth of penile relaxation length, the length of stretch and transverse and longitudinal diameters in the drug treatment group were higher than those in the physical treatment group, and the difference of growth values were statistically significant (all P<0.05). Conclusions:Both the negative pressure suction method and topical application of Testosterone cream are effective in the treatment of small and short penis after hypospadias surgery.However, Testosterone cream is difficult to obtain, and the treatment of negative pressure suction is simple, noninvasive, painless and free of adverse reactions.

Objective:To explore the effects of different doses of Ethephon on testes of male pups.Methods:Thirty-two 45-day-old healthy female Sprague-Dawley (SD) rats were randomly divided into the control group and the low dose, middle dose and high dose Ethephon groups by the random figure table.The female rats in the low dose, middle dose and high dose Ethephon groups were given 200, 400, and 800 mg/kg Ethephon solution, respectively.The control group was treated with 9 g/L saline.After the birth of the offspring, the mother rats were not administrated with any medications, and the male offspring rats were given Ethephon solution instead.Twelve offspring male rats were randomly selected from each group and killed at the age of 0, 14 and 28 days after birth.Fresh testicular tissues were stained with hematoxylin eosin (HE), and the morphological changes of testicular tissues were observed under light microscope.The apoptotic cells were labeled by terminal dexynucleotidyl transferase (TdT) mediated dUTP nick end labeling (TUNEL) method and the apoptosis index (AI) of spermatogenic cells was detected by fluorescence microscope.Results:(1) Compared with the newborn rats in the middle dose group, low dose group and control group, se-miniferous tubules in the newborn rats of the high dose group were slightly thicker, and seminiferous cells were arranged slightly in disorder.The AI of the newborn rats in high dose group was significantly higher than that in the control group (1.00±0.06 vs.0.41±0.03, P<0.01). The AI of the newborn rats in the middle dose group was not significantly different from that in the control group and the low dose group ( P>0.05). (2) The seminiferous tubules of the 14-day-old rats in the low dose, middle dose and high dose Ethephon groups were significantly thicker and arranged more loosely than those in the control group.Compared with the control group, there were very few seminiferous cells, which were arranged disorderly in the low dose, middle dose and high dose Ethephon groups.The AI of the 14-day-old rats in the low dose, middle dose and high dose Ethephon groups was (2.13±0.10), (2.18±0.10) and (3.90±0.23), respectively, which were significantly higher than that in the control group (1.00±0.02) ( F=2 508.36, P<0.01). There was no significant difference in the AI between the middle dose and low dose groups ( P>0.05). (3) Compared with the control group, the seminiferous tubules of the 28-day-old rats in the low dose, middle dose and high dose groups were significantly thicker and arranged much more loosely, and spermatogenic cells were even less and arranged in a severely disordered way.The AI of 28-day-old rats in the low dose group (5.52±0.13), the middle dose group (9.44±0.07) and the high dose group (14.56±0.27) was significantly higher than that in the control group (3.11±0.13) ( F=10 784.69, P<0.01). Conclusions:Ethephon can thicken the seminiferous tubules of newborn and young rats, cause the germ cells to arrange disorderly, promote the apoptosis of spermatogenic cells and reduce the ability of spermatogenesis.Moreover, a longer exposure of the rats to a higher concentration of Ethephon will result in more serious damage to testicular tissues.

To construct a eukaryotic expression plasmid containing the luciferase reporter gene (Fluc) to quickly detect apoptosis. Four amino acids, Asp-Glu-Val-Asp (DEVD), the recognize motif of Caspase-3, were introduced into the middle of the Fluc-C and N fragment. Meanwhile, four amino acids, Asp-Glu-Val-Gly (DEVG), were selected as a negative control. Subsequently, the recombinant gene was cloned into the N and C terminal end of the split intein, and named as pFluc-DEVD and pFluc-DEVG. Then the plasmids were transfected into cells and renilla luciferase was co-transfected in each sample as an internal control for transfection efficiency. Then the apoptosis level was detected by the double luciferase reporter gene and the Western blotting analysis. The results showed that when apoptosis occurred, the content of firefly luciferase expressed in the pFluc-DEVD plasmid transfected group was about 3 times higher than pFluc-DEVG plasmid transfected group. Furthermore, Western blotting detection indicated that the Fluc level was significantly increased in pFluc-DEVD transfected group when pre-treated by apoptosis stimulants. The activation degree of Caspase-3 was closely related to the expression of Fluc, and had a significant statistical difference. These results confirmed that firefly luciferase protein expressed by pFluc-DEVD plasmid can be cleaved by the intracellular Caspase-3 enzyme, and this plasmid can accurately reflect the cell apoptosis level, which provides a useful method for quantitative detection of apoptosis.
Apoptosis ; Genes, Reporter ; Luciferases, Firefly ; Transfection

Objective: To investigate the effect of Ethephon on the testis pathological structure and apoptosis of spermatogenic cell in offspring male rats. Methods: Twenty-four healthy female SD rats of 45 days old were randomly divided into control group, low-dose Ethephon group, medium-dose Ethephon group and high-dose Ethephon group according to body weight.The male rats of the same age were selected to mate with female rats.The rats were fed with Ethephon solution of different concentrations or 9 g/L saline every day, and they were continued to be fed with Ethephon during pregnancy and lactation.At the age of 7 days and 14 days, 10 offspring male rats were randomly selec-ted from each group and were put to death.The testicular tissue was stained with HE, and the morphological changes in the testis were observed with light microscope; the apoptotic cells were labeled with terminal deoxynucleotidyl transfe-rase dUTP nick-end labeling (TUNEL method) and the apoptosis index(AI) of testis spermatogenic cells was detected with fluorescence microscope. Results: At the age of 7 days, the testis internal structure of the control group developed well, and the spermatic tubules were neatly and compactly arranged.In the low-dose Ethephon group, the seminiferous tubules of the testis were slightly smaller and the spermatogenic cells were loosely arranged compared with the control group.In the medium-dose Ethephon group, the testis seminiferous tubules were slightly disordered and the cell gap increased.In the high-dose Ethephon group, the testis development was poor, the diameter of seminiferous tubules decreased significantly, and the spermatogenic cells arrangement was in disorder.There was no statistically significant difference in spermatogenic cell AI between the low-dose group [(0.54±0.10)%] and the control group[(0.53±0.09)%] (P>0.05), while the spermatogenic cell AI in the medium-dose Ethephon group [(0.63±0.11)%] and the high-dose Ethephon group [(0.81±0.06)%] were higher than that in the control group, thus there exists a statistically significant difference (all P<0.01). The spermatogenic cell AI in the low-dose Ethephon group [(0.54±0.10)%] was lower than that in the medium-dose Ethephon group [(0.63±0.11)%], and the difference was statistically significant (P<0.05). The spermatogenic cell AI in the medium-dose Ethephon group was higher than that in the high-dose Ethephon group, and the difference was statistically significant (P<0.01). At the age of 14 days, the spermatogenic cells AI in control group, low-dose Ethephon group, medium-dose Ethephon group and high-dose Ethephon group were (0.54±0.08)%, (0.65±0.11)%, (0.77±0.11)%, and (0.88±0.10)% respectively, and the spermatogenic cells AI in all groups increased gradually, in which the differences were statistically significant (all P<0.01). Conclusions Excessive dose of Ethephon can induce pathological changes in testicular tissue and increase the apoptosis of spermatogenic cells, resulting in low fertility of offspring rats.

T-cell internal antigen-1 (TIA-1) has roles in regulating alternative pre-mRNA splicing, mRNA translation, and stress granule (SG) formation in human cells. As an evolutionarily conserved response to environmental stress, SGs have been reported in various species. However, SG formation in chicken cells and the role of chicken TIA-1 (cTIA-1) in SG assembly has not been elucidated. In the present study, we cloned cTIA-1 and showed that it facilitates the assembly of canonical SGs in both human and chicken cells. Overexpression of the chicken prion-related domain (cPRD) of cTIA-1 that bore an N-terminal green fluorescent protein (GFP) tag (pntGFP-cPRD) or Flag tag (pFlag-cPRD) induced the production of typical SGs. However, C-terminal GFP-tagged cPRD induced notably large cytoplasmic granules that were devoid of endogenous G3BP1 and remained stable when exposed to cycloheximide, indicating that these were not typical SGs, and that the pntGFP tag influences cPRD localization. Finally, endogenous cTIA-1 was recruited to SGs in chicken cells and tissues under environmental stress. Taken together, our study provide evidence that cTIA-1 has a role in canonical SG formation in chicken cells and tissues. Our results also indicate that cPRD is necessary for SG aggregation.
Chickens ; Clone Cells ; Cycloheximide ; Cytoplasmic Granules ; Humans ; Protein Biosynthesis ; RNA Precursors ; RNA-Binding Proteins ; T-Lymphocytes

Total knee arthroplasty is an effective method for the treatment of end-stage knee osteoarthrosis, which can effectively relieve joint pain and reconstruct the integrity of the joint. Whether the posterior cruciate ligament should be preserved during surgery or not, which is still in dispute. In recent years, posterior cruciate-retaining and substituting total knee prostheses are both applied in clinical practice. Both domestic and international studies have shown that there are no significant difference in patient satisfaction, knee flexion, survival rate of the prosthesis and the main clinical manifestations between two prostheses. However, posterior cruciate-retaining total knee prosthesis is more consistent with the normal physiology and biomechanics of the human body. The gait is more balanced and proprioceptive when walking up and down the stairs, but when the joints are buckling, the femur is abnormal to move back to the tibia, resulting in abnormal motion. While posterior cruciate-substituting total knee prosthesis can correct severe deformity of the knee, and keep better balance between flexion and extension of the knee joint, but there is a potential complication of patellar clunk syndrome. Therefore, under the same conditions, the younger patients may prefer to chose posterior cruciate-retaining total knee prosthesis, while elder patients may prefer to chose posterior cruciate-substituting total knee prosthesis. This paper reviews the function of posterior cruciate ligament, as well as the advantages and disadvantages of two prostheses, so as to provide some references for clinic.

Objective To explore the interaction between tryptophan hydroxylase 2(TPH2) gene polymorphisms (rs4570625,rs11178997) and serotonin 1A receptor (5-HT1A) gene polymorpbisms (rs878567,rs1364043,rs6265) and the association with major depressive disorder (MDD) in a Chinese Han population.Methods The DNA isolated from peripheral blood samples of 288 MDD patients 288 healthy subjects was detected by single base primer extension assay (Snapshot).The generalized multifactor dimensionality reduction (GMDR) method was used to analyze the gene-gene interaction.Results Significant differences were found in the genotype (patients (TT:27,TA:152,AA:109),controls (TT:82,TA:105,AA:101),P＜0.01) and allele(patients (T:206,A:370),controls (T:269,A:307),P＜0.01) frequencies of rs1 1178997 within TPH2 between MDD patients and controls.Statistically,a greater risk of developing MDD was found in individuals with an rs1 1178997 A-allele(OR=1.574,95％CI=1.243-1.993).The interaction between TPH2 (rs4570625,rs1 1178997) and 5-HT1A (rs878567,rs1364043,rs6265) was considered as the best multi-locus model,and this showed a testing accuracy of 57.67％ and a CV consistency of 10/10.And this interaction had a significant effect on the risk of MDD (P=0.0107).Conclusion There may be an association between the interaction of TPH2 and 5-HT1A polymorphisms and MDD.