OBJECTIVE: To observe the neuroprotective effect of 6-shogaol (6-SH) in global cerebral ischemia/reperfusion injury (CIRI) following cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) in rats. METHODS: Computer-aided molecular docking was used to determine whether 6-SH could spontaneously bind to death-associated protein kinase 1 (DAPK1). SPF-grade male SD rats were randomly divided into a sham group (n = 5), a CPR group (n = 7), and a CPR+6-SH group (n = 7). The CPR group and CPR+6-SH group were further divided into 12-, 24-, and 48-hour subgroups based on observation time points. A rat model of global CIRI after CA-CPR was established by asphyxiation. In the sham group, only tracheal and vascular intubation was performed without asphyxia and CPR induction. The CPR group was intraperitoneally injected with 1 mL of normal saline immediately after successful modeling. The CPR+6-SH group received an intraperitoneal injection of 20 mg/kg 6-SH (1 mL) immediately after successful modeling, followed by administration every 12 hours until the endpoint. Neurological Deficit Score (NDS) was recorded at each time point after modeling. After completion of observation at each time point, rats were anesthetized and sacrificed, and brain tissue specimens were collected. Histopathological changes of neurons were observed under light microscopy after hematoxylin-eosin (HE) staining. Ultrastructural changes of hippocampal neurons and autophagy were observed by transmission electron microscopy (TEM). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect mRNA expression levels of DAPK1, vacuolar protein sorting 34 (VPS34), Beclin1, and microtubule-associated protein 1 light chain 3 (LC3) in brain tissues. Western blotting was used to detect protein expression levels of DAPK1, phosphorylated DAPK1 at serine 308 (p-DAPK1 ser308), VPS34, Beclin1, and LC3. Immunofluorescence was used to observe Beclin1 and LC3 expression in brain tissues under a fluorescence microscope. RESULTS: Molecular docking results indicated that 6-SH could spontaneously bind to DAPK1. Compared with the sham group, the NDS scores of the CPR group rats were significantly increased at all modeling time points; under light microscopy, disordered cell arrangement, widened intercellular spaces, and edema were observed in brain tissues, with pyknotic and necrotic nuclei in some areas; under TEM, mitochondria were markedly swollen with intact membranes, dissolved matrix, reduced or disappeared cristae, vacuolization, and increased autophagosomes. Compared with the CPR group, the NDS scores of the CPR+6-SH group rats were significantly decreased at all modeling time points; under light microscopy, local neuronal edema and widened perinuclear space were observed; under TEM, mitochondria were mostly mildly swollen with intact membranes, fewer autophagosomes, and alleviated injury. RT-qPCR results showed that compared with the sham group, mRNA expression levels of DAPK1, VPS34, Beclin1, and LC3 in brain tissues were significantly upregulated in all CPR subgroups, with the most pronounced changes at 24 hours. Compared with the CPR group, the CPR+6-SH group showed significantly lower mRNA expression of the above indicators at each time point [24 hours post-modeling (relative expression): DAPK1 mRNA: 3.41±0.68 vs. 4.48±0.62; VPS34 mRNA: 3.63±0.49 vs. 4.66±1.18; Beclin1 mRNA: 3.08±0.49 vs. 4.04±0.22; LC3 mRNA: 2.60±0.36 vs. 3.67±0.62; all P < 0.05]. Western blotting results showed that compared with the sham group, the protein expression levels of DAPK1, VPS34, Beclin1, and LC3 in all CPR subgroups were significantly increased, while the expression of p-DAPK1 ser308 was significantly decreased, with the most pronounced changes observed in the CPR 24-hour subgroup. Compared with the CPR group, the CPR+6-SH subgroups exhibited significantly reduced protein expression of DAPK1, VPS34, Beclin1, and LC3 [24-hour post-modeling: DAPK1/β-actin: 1.88±0.22 vs. 2.47±0.22; VPS34/β-actin: 2.55±0.06 vs. 3.46±0.05; Beclin1/β-actin: 2.12±0.03 vs. 2.87±0.03; LC3/β-actin: 2.03±0.24 vs. 3.17±0.23; all P < 0.05]. Conversely, the expression of p-DAPK1 ser308 was significantly upregulated in the CPR+6-SH group compared to the CPR group [24-hour post-modeling: p-DAPK1 ser308/β-actin: 0.40±0.02 vs. 0.20±0.07, P < 0.05]. Under the fluorescence microscope, fluorescence intensities of Beclin1 and LC3 in the CPR 24-hour group were significantly higher than those in the sham 24-hour group; compared with the CPR 24-hour group, the CPR+6-SH 24-hour group showed significantly reduced fluorescence intensities of Beclin1 and LC3. CONCLUSION 6-SH inhibited the expression of DAPK1, alleviated excessive autophagy after global CIRI following CA-CPR in rats, and exerted neuroprotective effects. The mechanism may be related to phosphorylation at the DAPK1 ser308 site.
Animals ; Rats, Sprague-Dawley ; Male ; Rats ; Cardiopulmonary Resuscitation ; Autophagy/drug effects* ; Heart Arrest/therapy* ; Death-Associated Protein Kinases/metabolism* ; Reperfusion Injury/metabolism* ; Disease Models, Animal ; Neuroprotective Agents/pharmacology* ; Brain Ischemia/metabolism*

OBJECTIVE To establish a quantitative analysis model for the contents of tussilagone， moisture， ethanol-soluble extract and total ash in Farfarae Flos based on near-infrared spectroscopy （NIRS）， providing a new idea for the rapid quality evaluation of Farfarae Flos and its preparations. METHODS Referring to the 2020 edition of the Chinese Pharmacopoeia， the contents of the main quality control indexes tussilagone， moisture， ethanol-soluble extract and total ash in 130 batches of Farfarae Flos from 19 producing areas were determined by HPLC， drying method， hot dip method and ash assay， respectively. The NIRS data information of the medicinal herbs of Farfarae Flos was collected， and then NIRS combined with the partial least squares method was used to establish the individual quantitative analysis models of the above quality control indexes in the samples， and the predictive model of the NIRS content was obtained after sample validation with validation set. RESULTS The range for the contents of tussilagone， moisture， ethanol-soluble extract and total ash in 130 batches of Farfarae Flos were 0.051 4%-0.103 5%， 7.75%-10.93%， 20.17%-31.12%， and 7.68%-12.10%， respectively. The internal cross-validation coefficients of determination （R2） of the established models for the quantitative analysis of tussilagone， moisture， ethanol-soluble extract and total ash in Farfarae Flos were 0.985 8， 0.968 4， 0.973 4， 0.988 0， respectively； the root mean square errors of calibration （RMSEC） were 0.001 54， 0.187， 0.478， 0.127， respectively； the root mean square errors of prediction （RMSEP） were 0.001 81， 0.212， 0.543， 0.149， respectively； RMSEP/RMSEC were 1.175 3， 1.133 7， 1.136 0 and 1.173 2， respectively， which were all within a reasonable range （1＜RMSEP/RMSEC≤1.2）. The mean absolute errors between the true and model-predicted values of the above four quality control indexes in the validation set of samples were －0.000 36， 0.061 43， 0.144 00， and 0.010 43， respectively，and the mean predicted recoveries were 99.65%， 100.72%，100.66%， and 100.15%， respectively. CONCLUSIONS The established NIRS quantitative analysis model has high stability and reliable results， which can be used for the rapid batch prediction of the content of relevant quality control indexes in Farfarae Flos.

AIM:To establish a mouse model of chronic obstructive pulmonary disease(COPD)induced by cigarette smoke(CS)exposure combined with polyinosinic-polycytidylic acid(Poly I:C)nasal drip,and to investigate the mechanism of airway epithelial barrier injury in COPD.METHODS:(1)Ninety-six male BALB/c mice were randomly divided into control group,CS group,Poly I:C group,and CS+Poly I:C group(n=24).The model was established from week 1 to week 8,with pulmonary function tested every 4 weeks.Six mice from each group were sacrificed at the end of weeks 4,8,16,and 24.Changes in minute volume(MV),enhanced pause(Penh),mean linear intercept(MLI)and bronchial wall thickness(BWT)were observed.The protein levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),zonula occludens-1(ZO-1)and E-cadherin(E-Cad)in the lung were detected.(2)Human bronchial epithe-lial BEAS-2B cells were stimulated with CS extract(CSE)combined with Poly I:C for 24 h,and then the protein levels of occludin(Occ),ZO-1,and phosphorylated epidermal growth factor receptor(EGFR),P38 and extracellular signal-regu-lated kinase(ERK)1/2 were analyzed.RESULTS:(1)Compared with control group,at the 8th week,the mice in CS and CS+Poly I:C groups showed typical pathological changes in lung tissues,including significant inflammatory cell infil-tration,alveolar cavity expansion,alveolar wall rupture and fusion,and airway wall thickening.The Penh,BWT,MLI,and lung IL-1β and TNF-α levels were significantly increased(P<0.05 or P<0.01),while MV and lung ZO-1 and E-Cad levels were remarkably decreased(P<0.05 or P<0.01).By the 24th week,these pathological changes remained relative-ly stable in CS+Poly I:C group.(2)Compared with control group,CSE and its combination with Poly I:C dramatically in-duced a reduction in ZO-1 and Occ protein expression in BEAS-2B cells(P<0.05 or P<0.01),and increased the levels of phosphorylated EGFR,P38 and ERK1/2(P<0.01).The effects in CSE combined with Poly I:C group were considerably superior to those in CSE or Poly I:C group alone.CONCLUSION:Poly I:C can enhance the pathological changes and airway epithelial barrier damage induced by CS in a mouse model of COPD,which may be related to the activation of EGFR/ERK/P38 signaling pathway.