Human carboxylesterase 2A (hCES2A) plays pivotal roles in prodrug activation and hydrolytic metabolism of ester-bearing chemicals. Targeted inhibition of intestinal hCES2A represents a feasible strategy to mitigate irinotecan-triggered gut toxicity (ITGT), but the orally active, selective, and efficacious hCES2A inhibitors are rarely reported. Here, a novel drug-like hCES2A inhibitor was developed via three rounds of structure-based drug design (SBDD) and structural optimization. Initially, donepezil was identified as a moderate hCES2A inhibitor from 2000 US Food and Drug Administration (FDA)-approved drugs. Following two rounds of SBDD and structural optimization, a donepezil derivative (B7) was identified as a strong reversible hCES2A inhibitor. Subsequently, nine B7 carbamates were rationally designed, synthesized and biologically assayed. Among all synthesized carbamates, C3 showed the most potent time-dependent inhibition on hCES2A (IC₅₀ = 0.56 nmol/L), excellent specificity and favorable drug-like properties. C3 could covalently modify the catalytic serine of hCES2A with high selectivity, while this agent also showed favorable safety profiles, high intestinal exposure, and impressive effects for ameliorating ITGT in both human intestinal organoids and tumor-bearing mice. Collectively, this study showcases a rational strategy for developing drug-like and serine-targeting covalent inhibitors against target serine hydrolase(s), while C3 emerges as a promising orally active drug candidate for ameliorating ITGT.

OBJECTIVES: To explore the effects of acupuncture combined with rehabilitation training for promoting transdifferentiation of astrocytes into neurons in mice after cerebral ischemia. METHODS: Male C57/BL6J mice were subjected to intracerebral microinjection of an adeno-associated virus carrying the GFAP promoter for NeuroD1 and Ngn2 overexpression in the astrocytes, followed 3 or 12 days later by electrocoagulation of the distal middle cerebral artery. After modeling, the mice were randomly divided into model group without interventions and intervention group treated with electroacupuncture at the acupoints Baihui (GV20), left Hegu (LI4), Neiguan (PC6), Zusanli (ST36), and Yanglingquan (GB34) 24 h after surgery. The mice in the intervention group were housed individually in cages with running wheels, and their activity was recorded every 24 h. Neurological function scores of the mice were assessed on the 1st, 14th, and 21st days after modeling. Transdifferentiation of astrocytes in the target brain regions was observed using double immunofluorescence staining. RESULTS: Compared with those in the model group, the mice receiving eletroacupuncture and rehabilitation training showed significant improvement of neurological deficits at 14 and 21 days after modeling. The GFAP promoter of the AAV2/5 vector specifically labeled the local astrocytes, and compared with that that in the model group, the number of AAV-positive cells colabeled with the neuronal marker DCX significantly increased after 14 days of electroacupuncture and rehabilitation intervention, and the number of AAV-positive cells colabeled with the neuronal marker NeuN significantly increased after 21 days of intervention. CONCLUSIONS In mice with cerebral ischemia, electroacupuncture and rehabilitation training can promote transdifferentiation of astrocytes into neurons in the ischemic brain region, and the efficiency of transdifferentiation is positively correlated with the improvement of motor function.
Animals ; Electroacupuncture ; Astrocytes/cytology* ; Cell Transdifferentiation ; Male ; Mice, Inbred C57BL ; Brain Ischemia/physiopathology* ; Mice ; Neurons/cytology* ; Doublecortin Protein

Objective To modify the method for establishing mouse models of middle cerebral artery occlusion(MCAO)-induced focal cerebral ischemia using electrocoagulation.Methods Forty-six C57/BL6J male mice were divided into MCAO model group(n=34)and sham-operated group(n=12).In the model group,MCAO was induced by permanent coagulation of the right middle cerebral artery(MCA)using a coagulator,and cerebral blood flow perfusion was monitored before and at 20 min and 1 day after modeling.Neurological deficits of the mice at 1,7,and 14 days after modeling were evaluated using Longa score,mNSS score,beam walking test,cylinder test and corner test.TTC staining was used to measure the cerebral infarct size,and Western blotting was performed to detect the expressions of BDNF,GFAP and DCX proteins in the ischemic cortex.Results The mice in the model group showed significantly reduced cerebral blood flow in the MCA on the ischemic side and obvious neurological deficits with increased forelimb use asymmetry on days 1,7 and 14 after modeling(P<0.05).In the cerebral cortex on the ischemic side of the model mice,the expressions of GFAP and DCX increased significantly at 1,7,and 14 days(P<0.05)and the expression of BDNF increased at 1 day after modeling ischemia(P<0.05).Conclusion We successfully prepared mouse models of MCAO using a modified method by changing the electrocoagulation location from the distal location of the junction between the MCA and the inferior cerebral vein to a 2 mm segment medial to the junction between the MCA and the olfactory bundle.

Objective To modify the method for establishing mouse models of middle cerebral artery occlusion(MCAO)-induced focal cerebral ischemia using electrocoagulation.Methods Forty-six C57/BL6J male mice were divided into MCAO model group(n=34)and sham-operated group(n=12).In the model group,MCAO was induced by permanent coagulation of the right middle cerebral artery(MCA)using a coagulator,and cerebral blood flow perfusion was monitored before and at 20 min and 1 day after modeling.Neurological deficits of the mice at 1,7,and 14 days after modeling were evaluated using Longa score,mNSS score,beam walking test,cylinder test and corner test.TTC staining was used to measure the cerebral infarct size,and Western blotting was performed to detect the expressions of BDNF,GFAP and DCX proteins in the ischemic cortex.Results The mice in the model group showed significantly reduced cerebral blood flow in the MCA on the ischemic side and obvious neurological deficits with increased forelimb use asymmetry on days 1,7 and 14 after modeling(P<0.05).In the cerebral cortex on the ischemic side of the model mice,the expressions of GFAP and DCX increased significantly at 1,7,and 14 days(P<0.05)and the expression of BDNF increased at 1 day after modeling ischemia(P<0.05).Conclusion We successfully prepared mouse models of MCAO using a modified method by changing the electrocoagulation location from the distal location of the junction between the MCA and the inferior cerebral vein to a 2 mm segment medial to the junction between the MCA and the olfactory bundle.

Objective To explore the effect of simvastatin on the proliferation,apoptosis and protein expressions of keloid fibroblasts under normoxia,hypoxia or TGF-β1 treatment.Methods Keloid fibroblasts (KFs) were isolated by explants culture method.KFs were treated with different concentrations of simvastatin under normoxia or hypoxia (2％ O2) for 24 h and 48 h.The effects of simvastatin on cell proliferation were detected by CCK-8.Flow cytometer was used to detect the apoptosis of KFs treated with 10 μ mol/L simvastatin for 24 h or 48 h under normoxia,hypoxia or 10 ng/ml TGF-β1 treatment.Then the expressions of keloid-related proteins were analyzed by Western Blot.Results It showed that simvastatin could inhibit the proliferation of KFs in a concentration-and time-dependent manner with the concentration range of 10-500 μ mol/L for 24 h and 0.1-500 μ mol/L for 48 h.This inhibitory effect could be significantly enhanced when cells were incubated under hypoxia for 48h with 10-500 μ mol/L simvastatin.10 μ mol/L simvastatin could not influence the apoptosis of KFs under normoxia or TGF-β1 treatment,neither incubated for 24 h nor 48 h.When incubated under hypoxia,10 μ mol/L simvastatin could significantly induce the apoptosis of KFs,with the rate of 155.6％ for 24 h and 478.8％ for 48 h,compared with no-drug control.There are no significant influences on the expression of type Ⅰ collagen,CTGF or TIMP-1 when KFs were treated with 10 μ mol/L simvastatin under normoxia for 48 h.When incubated with 10 ng/ml TGF-β1 together with 10 μmol/L simvastatin for 48 h,the expression of CTGF was significantly inhibited.KFs treated with 10 μ mol/L simvastatin under hypoxia for 48 h showed a significant decrease of type Ⅰ collagen and CTGF,and a significant increase of TIMP-1.Conclusions Simvastatin has different effects on the proliferation,apoptosis and protein expressions of KFs in a dosedependent manner under different conditions.The effects are enhanced under hypoxia.

Objective To explore the effect of simvastatin on the proliferation,apoptosis and protein expressions of keloid fibroblasts under normoxia,hypoxia or TGF-β1 treatment.Methods Keloid fibroblasts (KFs) were isolated by explants culture method.KFs were treated with different concentrations of simvastatin under normoxia or hypoxia (2％ O2) for 24 h and 48 h.The effects of simvastatin on cell proliferation were detected by CCK-8.Flow cytometer was used to detect the apoptosis of KFs treated with 10 μ mol/L simvastatin for 24 h or 48 h under normoxia,hypoxia or 10 ng/ml TGF-β1 treatment.Then the expressions of keloid-related proteins were analyzed by Western Blot.Results It showed that simvastatin could inhibit the proliferation of KFs in a concentration-and time-dependent manner with the concentration range of 10-500 μ mol/L for 24 h and 0.1-500 μ mol/L for 48 h.This inhibitory effect could be significantly enhanced when cells were incubated under hypoxia for 48h with 10-500 μ mol/L simvastatin.10 μ mol/L simvastatin could not influence the apoptosis of KFs under normoxia or TGF-β1 treatment,neither incubated for 24 h nor 48 h.When incubated under hypoxia,10 μ mol/L simvastatin could significantly induce the apoptosis of KFs,with the rate of 155.6％ for 24 h and 478.8％ for 48 h,compared with no-drug control.There are no significant influences on the expression of type Ⅰ collagen,CTGF or TIMP-1 when KFs were treated with 10 μ mol/L simvastatin under normoxia for 48 h.When incubated with 10 ng/ml TGF-β1 together with 10 μmol/L simvastatin for 48 h,the expression of CTGF was significantly inhibited.KFs treated with 10 μ mol/L simvastatin under hypoxia for 48 h showed a significant decrease of type Ⅰ collagen and CTGF,and a significant increase of TIMP-1.Conclusions Simvastatin has different effects on the proliferation,apoptosis and protein expressions of KFs in a dosedependent manner under different conditions.The effects are enhanced under hypoxia.

OBJECTIVETo investigate the statement on randomized controlled trials on Wenxin granule for treatment of atrial fibrillation and to judge whether those trials could offer high quality evidence or not, thus improve design level and quality.

METHODRCTs were searched from home and abroad about atrial fibrillation treated with Wenxin granule, which reported before October, 2010. Jadad scale and CONSORT statement were used.

RESULTThere were 66 RCTs retrieved that met inclusion criteria. Using Jadad rating scale, only 2 literatures gain score 4 and 1 literature gains score 3, 54 literatures gain score 2, 7 literatures gain score 1, 2 literatures gain score 0. Only 2 literatures described random number table as the method of grouping. None of the RCTs was reported the allocation concealment. Only 1 literature was used blinding. Fifty-nine literatures were mentioned the lost to follow-up conditions. According to the CONSORT standards, only six literatures (9.1%) mentioned the method of generating the random sequence. Four literatures (6.1%) were quasi-random. Nineteen literatures (28.8%) had inclusion criteria. Six literatures (9.1%) had the follow-up record. Fifty one literatures (77.3%) described the adverse events. None had the estimation of the sample size, intention-to-treat analysis and stratified analysis. None had the ethical approval or informed consent.

CONCLUSIONThe quality of clinical trials of Wenxin granule in treating atrial fibrillation needs to be improved.