ObjectiveThis paper aims to investigate the differential mechanisms underlying the staged therapeutic effects of Qijia Rougan formula on liver fibrosis using proteomic technology. MethodsThe staged rat model of liver fibrosis was established by subcutaneous injection of carbon tetrachloride （CCl₄） and olive oil. One hundred and four SD rats were randomized into thirteen groups：a normal group，a two-week model group，a four-week model group，a six-week model group，an eight-week model group，a two-week Qijia Rougan formula group，a four-week Qijia Rougan formula group，a six-week Qijia Rougan formula group，an eight-week Qijia Rougan formula group，a two-week compound Biejia Ruangan tablet group，a four-week Compound Biejia Ruangan Tablet group，a six-week Compound Biejia Ruangan Tablet group，and an eight-week compound Biejia Ruangan tablet group. After two weeks of drug intervention，liver tissue and abdominal aortic blood samples were collected from the rats for testing. Hematoxylin-eosin （HE） staining，Masson staining，and Picro Sirius red staining were used to observe pathological damage and collagen fiber deposition in liver tissues. Immunohistochemistry （IHC） was employed to detect the contents of fibrosis markers in liver tissues. The contents of liver function indicators in the serum were measured using a fully automated biochemical analyzer，and the levels of liver fibrosis indicators in the serum were assessed by enzyme-linked immunosorbent assay （ELISA）. Liver tissues from the normal group，each model group，and each Qijia Rougan formula group were subjected to label-free quantitative proteomic analysis to identify differential proteins among the groups，with key proteins validated by Western blot. Finally，bioinformatics analysis was performed on the differential proteins. Results（1） The staged rat model of liver fibrosis constructed with CCl₄ and olive oil showed pathological results at the 2^nd，4^th，6^th，and 8^th weeks of modeling that were consistent with the Metavir standards for the F1，F2，F3，and F4 stages. Compared with those in the normal control group，the protein expressions of α-smooth muscle actin （α-SMA） and Collagen Ⅰ were significantly increased in each stage （P<0.05）. The levels of liver function indicators in the serum，including alanine aminotransferase （ALT），aspartate aminotransferase （AST），alkaline phosphatase （ALP），direct bilirubin （DBIL），and total bilirubin （TBil） in each model group，were significantly elevated in each stage （P<0.01）. The levels of liver fibrosis indicators in the serum，including procollagen Ⅲ peptide （PⅢP），type Ⅳ collagen（Ⅳ-C），hyaluronic acid （HA），and laminin （LN） in each model group，were significantly increased in each stage （P<0.05，P<0.01）. This study successfully established a staged rat model of liver fibrosis. （2） Compared with the model groups at each stage，the administration groups showed a reduction in hepatocyte ballooning degeneration，a more orderly arrangement of hepatocytes，and a decrease of inflammatory cell infiltration. The blue-stained collagen fibers became significantly thinner and finer，with reduced and narrowed fibrous septa. The areas of collagen fibers and Picro Sirius red staining were reduced （P<0.05）. The positive areas of α-SMA and Collagen Ⅰ expression were significantly decreased （P<0.05）. The levels of ALT，AST，ALP，DBIL，and TBil in the rats of the model groups at each stage were significantly reduced （P<0.05，P<0.01）. The levels of PⅢP，Ⅳ-C，HA，and LN in the rats of the model groups at each stage were significantly decreased （P<0.05）. Among these，the improvements in all indicators were most significant in the F3 stage （P<0.01）.（3） The proteomic results show that a total of 165 differential proteins exhibit a callback trend when comparing the model groups at four stages with the normal group，and when comparing the Qijia Rougan formula group with the model group. Western blot analysis reveals that the levels of NAD（P）H：quinone oxidoreductase 1 （NQO1），mitogen-activated protein kinase 1 （MAPK1），arginase 1 （Arg1），and glutathione S-transferase α1 （GSTA1） were consistent with the proteomic results. Bioinformatics results reveal that 165 differentially expressed proteins are enriched in multiple signaling pathways. Notably，signaling pathways such as drug metabolism-cytochrome P450，arginine biosynthesis，and the peroxisome proliferator-activated receptor （PPAR） signaling pathway were found to be closely associated with liver fibrosis，suggesting that the Qijia Rougan formula may exert its staged regulatory effects on liver fibrosis by regulating these pathways. ConclusionThe Qijia Rougan formula may achieve staged regulation of liver fibrosis by regulating drug metabolism-cytochrome P450，arginine biosynthesis，and the PPAR signaling pathway.

Objective To investigate the effect of sulforaphane(SFN)on the expression of apoptosis-related proteins(caspase-3 and caspase-9),in brain tissue of rats with acute carbon monoxide poisoning(ACOP),and to explore the molecular mechanism underlying its intervention in ACOP-induced brain injury.Methods The healthy male Sprague-Dawley(SD)rats were randomly assigned to three groups:normal control(NC)group,ACOP group,and SFN group,with 36 rats in each group.An ACOP animal model was established by exposing the rats to carbon monoxide(CO)in a hyperbaric oxygen chamber,while the rats in the NC group were allowed to breathe fresh air.The SFN group received an intraperitoneal injection of SFN 20 mg/kg within 2 hours after poisoning,once daily,until euthanasia.The NC and ACOP groups were injected with an equivalent volume of saline.Rats from each group were sacrificed on days 1,3,and 7 of the intervention to collect brain tissue,hematoxylin-eosin(HE)staining was performed to assess pathological damage in the brain tissue;Nissl staining was used to examine neuronal pathological changes;Immunohistochemistry was employed to detect the positive expression of caspase-3 and caspase-9 in the cortical region of the brain.Western blotting and quantitative reverse transcription-polymerase chain reaction(qRT-PCR)were conducted to measure the protein and mRNA expression of caspase-3 and caspase-9 in the brain tissue.Results After CO poisoning,brain tissue damage in the ACOP group progressively worsened,with a gradual decrease in the number of Nissl bodies and a gradual increase in the number of positive cells for caspase-3 and caspase-9 in the cortical region of the brain.The protein and mRNA expression of caspase-3 and caspase-9 in the brain tissue also gradually increased.Compared with the NC group at the same time point,the differences were statistically significant[Nissl bodies:69.33±0.94 vs.91.33±1.25;caspase-3 positive expression(A value):0.149±0.003 vs.0.113±0.004;caspase-9 positive expression(A value):0.178±0.002 vs.0.111±0.010;caspase-3 protein(caspase-3/GAPDH):1.634±0.045 vs.0.844±0.021;caspase-9 protein(caspase-9/GAPDH):1.754±0.024 vs.0.811±0.053;caspase-3 mRNA(2-ΔΔCt):1.718±0.052 vs.1;caspase-9 mRNA(2-ΔΔCt):1.722±0.066 vs.1,all P<0.05).Compared with the ACOP group at the same time point,the brain tissue damage in the SFN group improved,with a significant increase in the number of Nissl bodies(84.67±1.53 vs.69.33±0.94,P<0.05).The number of positive cells for caspase-3 and caspase-9 in the cortical region of the brain decreased significantly(A value:0.126±0.002 vs.0.149±0.003,0.127±0.002 vs.0.178±0.002,both P<0.05).The protein and mRNA expression of caspase-3 and caspase-9 in the brain tissue were significantly reduced[caspase-3 protein(caspase-3/GAPDH):0.999±0.037 vs.1.634±0.045;caspase-9 protein(caspase-9/GAPDH):0.993±0.040 vs.1.754±0.024;caspase-3 mRNA(2-ΔΔCt):1.120±0.059 vs.1.718±0.052;caspase-9 mRNA(2-ΔΔCt):0.520±0.045 vs.1.722±0.066,all P<0.05].Conclusion SFN partially attenuated ACOP-induced brain injury in rats,potentially by downregulating both protein and mRNA expression of caspase-3 and caspase-9,thereby reducing cellular apoptosis.

Objective:To explore the neuroprotective effect and molecular mechanism of sulforaphane (SFN) on acute carbon monoxide poisoning (ACOP) in rats.Methods:A total of 135 healthy adult male Sprague-Dawley (SD) rats were randomly divided into normal control group, ACOP model group, and SFN intervention group, with 45 rats in each group. The ACOP animal model was reproduced using carbon monoxide (CO) inhalation in a hyperbaric oxygen chamber, while the normal control group was allowed to breathe fresh air freely. The rats in the SFN intervention group received intraperitoneal injection of SFN at a dose of 20 mg/kg once daily starting 2 hours after CO poisoning and continuing until euthanasia. The normal control group and the ACOP model group received equivalent volume of saline injection. Three rats from each group were sacrificed 1 day after intervention to observe the changes in the ultrastructure of neuronal mitochondria in brain tissues under transmission electron microscopy. Six rats from each group were evaluated for cognitive function using neurobehavioral test 7 days after intervention. Brain tissues of 6 rats in each group were collected 1, 3, and 7 days after intervention, and the expressions of phosphorylated-adenosine monophosphate-activated protein kinase (p-AMPK), mitofusin 2 (MFN2), and dynamin-related protein 1 (DRP1) were detected using immunohistochemistry staining and Western blotting. Linear regression analysis was performed to assess the correlations between the expression levels of above proteins.Results:In the normal control group, the rats did not exhibit any abnormalities in cognitive function or the ultrastructure of neuronal mitochondria in brain tissues. ACOP induced cognitive impairment and ultrastructural injury to neuronal mitochondria in rats. However, SFN significantly improved cognitive function in poisoned rats and mitigated the extent of neuronal mitochondrial damage. Over poisoning time, the expression levels of p-AMPK and MFN2 in the brain tissues of ACOP rats were gradually decreased, while the expression level of DRP1 was gradually increased. Compared with the normal control group, the ACOP model group showed significant differences in the expressions of p-AMPK, MFN2, and DRP1. After SFN intervention, the expression levels of above proteins were significantly reversed. Compared with the ACOP model group, the SFN intervention group exhibited a marked increase in the expressions of p-AMPK and MFN2 [p-AMPK positive expression ( A value): 0.226±0.003 vs. 0.177±0.033, p-AMPK protein (p-AMPK/GAPDH): 1.41±0.05 vs. 0.89±0.05, MFN2 positive expression ( A value): 0.241±0.004 vs. 0.165±0.007, MFN2 protein (MFN2/GAPDH): 1.33±0.04 vs. 0.79±0.03, all P < 0.05], along with a significant decrease in DRP1 expression [DRP1 positive expression ( A value): 0.103±0.002 vs. 0.214±0.011, DRP1 protein (DRP1/GAPDH): 1.00±0.03 vs. 1.50±0.03, both P < 0.05]. Linear regression analysis revealed a strong negative linear correlation between DRP1 protein expression and MFN2, p-AMPK protein expressions ( R2 values were 0.977 and 0.971, both P < 0.01), and a positive linear correlation between p-AMPK protein expression and MFN2 protein expression ( R2 = 0.985, P < 0.01). Conclusion:SFN can help maintain neuronal mitochondrial homeostasis by activating the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, thereby alleviating neuronal injury caused by ACOP.

Objective To investigate the effect of sulforaphane(SFN)on the expression of apoptosis-related proteins(caspase-3 and caspase-9),in brain tissue of rats with acute carbon monoxide poisoning(ACOP),and to explore the molecular mechanism underlying its intervention in ACOP-induced brain injury.Methods The healthy male Sprague-Dawley(SD)rats were randomly assigned to three groups:normal control(NC)group,ACOP group,and SFN group,with 36 rats in each group.An ACOP animal model was established by exposing the rats to carbon monoxide(CO)in a hyperbaric oxygen chamber,while the rats in the NC group were allowed to breathe fresh air.The SFN group received an intraperitoneal injection of SFN 20 mg/kg within 2 hours after poisoning,once daily,until euthanasia.The NC and ACOP groups were injected with an equivalent volume of saline.Rats from each group were sacrificed on days 1,3,and 7 of the intervention to collect brain tissue,hematoxylin-eosin(HE)staining was performed to assess pathological damage in the brain tissue;Nissl staining was used to examine neuronal pathological changes;Immunohistochemistry was employed to detect the positive expression of caspase-3 and caspase-9 in the cortical region of the brain.Western blotting and quantitative reverse transcription-polymerase chain reaction(qRT-PCR)were conducted to measure the protein and mRNA expression of caspase-3 and caspase-9 in the brain tissue.Results After CO poisoning,brain tissue damage in the ACOP group progressively worsened,with a gradual decrease in the number of Nissl bodies and a gradual increase in the number of positive cells for caspase-3 and caspase-9 in the cortical region of the brain.The protein and mRNA expression of caspase-3 and caspase-9 in the brain tissue also gradually increased.Compared with the NC group at the same time point,the differences were statistically significant[Nissl bodies:69.33±0.94 vs.91.33±1.25;caspase-3 positive expression(A value):0.149±0.003 vs.0.113±0.004;caspase-9 positive expression(A value):0.178±0.002 vs.0.111±0.010;caspase-3 protein(caspase-3/GAPDH):1.634±0.045 vs.0.844±0.021;caspase-9 protein(caspase-9/GAPDH):1.754±0.024 vs.0.811±0.053;caspase-3 mRNA(2-ΔΔCt):1.718±0.052 vs.1;caspase-9 mRNA(2-ΔΔCt):1.722±0.066 vs.1,all P<0.05).Compared with the ACOP group at the same time point,the brain tissue damage in the SFN group improved,with a significant increase in the number of Nissl bodies(84.67±1.53 vs.69.33±0.94,P<0.05).The number of positive cells for caspase-3 and caspase-9 in the cortical region of the brain decreased significantly(A value:0.126±0.002 vs.0.149±0.003,0.127±0.002 vs.0.178±0.002,both P<0.05).The protein and mRNA expression of caspase-3 and caspase-9 in the brain tissue were significantly reduced[caspase-3 protein(caspase-3/GAPDH):0.999±0.037 vs.1.634±0.045;caspase-9 protein(caspase-9/GAPDH):0.993±0.040 vs.1.754±0.024;caspase-3 mRNA(2-ΔΔCt):1.120±0.059 vs.1.718±0.052;caspase-9 mRNA(2-ΔΔCt):0.520±0.045 vs.1.722±0.066,all P<0.05].Conclusion SFN partially attenuated ACOP-induced brain injury in rats,potentially by downregulating both protein and mRNA expression of caspase-3 and caspase-9,thereby reducing cellular apoptosis.

Objective:To observe the effect of early temperature control on the prognosis of brain injury patients after severe carbon monoxide poisoning (COP).Methods:A total of 277 patients hospitalized with severe COP were randomly divided into a fever group ( n=78), a normal temperature group ( n=113) and a mild hypothermia group ( n=86). All were given hyperbaric oxygen therapy and any necessary supportive treatment. The mild hypothermia group were kept in a room at 34 to 35℃. Evaluation was with the Glasgow Coma Scale (GCS), version II of the Acute Physiology and Chronic Health Evaluation (APACHE), the Hasegawa dementia scale (HDS) and the mini mental state examination (MMSE). The incidence of delayed encephalopathy (DEACMP) and mortality were compared among the three groups. The bispectral index (BIS) and neuron-specific enolase (NSE) levels were correlated with DEACMP. Results:After the treatments, improvement was observed in multiple indexes of all three groups compared with before the treatment. Compared with the fever group, the average GCS of the mild hypothermia group was significantly higher on the 2nd, 4th, 8th and 31st day after the intervention. It was significantly higher than the normal temperature group′s averages on the 4th, 8th and 31st day. The average APACHE scores of the normal temperature and the mild hypothermia groups were significantly lower than the fever group′s average, with that of the mild hypothermia group significantly lower than that of the normal group. The average HDS scores of the normal temperature and mild hypothermia groups were significantly higher than the fever group′s average, with that of the mild hypothermia group significantly higher than that of the normal group. The average MMSE score of the mild hypothermia group was significantly improved after 7 days, one month and three months of treatment. That of the normal group showed significant improvement after one and three months, but the mild hypothermia group′s averages were superior. Compared with the fever group, the average BIS score of the mild hypothermia group was significantly better after one, three and seven days, and one month. This was true for the normal group beyond three days after the intervention. The average NSE concentration of the normal group after 7 days and one month was significantly lower than that of the fever group. For the mild hypothermia group this was true after only 3 days. Compared with the other two groups, the average coma time, incidence of DEACMP and nervous system injury were significantly lower in the hypothermia group. The average GCS, BIS and NSE values were closely related to the occurrence of DEACMP.Conclusions:Early temperature control can significantly reduce the severity of brain injury after COP and reduce the incidence of neurological sequelae. Early dynamic detection of GCS, NSE concentration and BIS is of great significance for predicting the incidence of DEACMP.