| Objectives:Through literature exploration,we sorted out the connection between Nrf2 signaling pathway and AD and the mechanism of Huang Lian Jiedu Decoction in treating AD,and hypothesized that Huang Lian Jiedu Decoction might treat AD through Nrf2 signaling pathway.To investigate the hidden mechanism of Huanglian Jiedu Decoction in treating AD,and to offer a new experiment foundation for treating AD with Huanglian Jiedu Decoction.To evaluate the efficacy,safety and effects of oxidative stress indexes of Huanglian Jiedu Decoction in the treatment of mild to moderate AD patients through clinical studies,to confirm that Huanglian Jiedu Decoction is an effective treatment protocol for AD,and to explore whether the antioxidant effects of Huanglian Jiedu Decoction are consistent with the results of experimental studies.Methods:1.Experimental research methods:In this study,60 9-month-old male AD model mice bred and identified as 3x-Tg triple-transgenic and 12 9-month-old background mice of this triple-transgenic mouse C57BL/6J were used for the study.Twelve C57BL/6J rats at 9 months old were treated as normal subjects,and 60 identical 3x-Tg AD rats were randomized to Huanglian Jiedu Decoction(HLJDT-L,M,H),and Donepezil.The control group and the model group were given 0.9%saline gavage,and the remaining groups were given the corresponding doses of Donepezil and Huanglian Jiedu Decoction by gavage.The gavage doses of Huanglian Jiedu Decoction were1950 mg/kg/d,3900 mg/kg/d and 7800 mg/kg/d for low,medium and high gavage,respectively,and the dose of Donepezil was 0.65mg/kg/d.The anxiety state and learning and memory ability of each group were evaluated by using the absentminded field test,new object recognition test and Morris water maze test for 1month,and HE The expression levels of A β 1-42 in the hippocampus of each group of mice were detected by ELISA;the expression levels of MDA,SOD and GSH in the hippocampus of each group of mice were detected by each kit,and the expression levels of ROS in the hippocampus of each group of mice were detected by flow cytometry;the expression levels of Nrf2 in the hippocampus of each group of mice were detected by immunofluorescence.Using immunofluorescent method,Nrf2 was measured in rat hippocampus,and Nrf2 was measured by Western blot;the expression levels of HO-1 and NQO1,the downstream proteins of Nrf2 signaling pathway in the hippocampus of mice were detected by Western blot;the ALT,AST,BUN and Cr of mice before and after gavage were detected by biochemical assay.2.Clinical research methods:Altogether 60 eligible cases were selected to be randomly assigned to the control group,30 cases to observe group.The control group was treated with donepezil and the observation group was treated with donepezil plus Huanglian Jiedu Decoction for 12 weeks,with 4 weeks as a course of treatment.The baseline conditions of the two groups before treatment were compared to assess whether they were comparable;the MMSE scale was used to compare the cognitive function,the ADL scale to compare the ability of daily living,the NPI scale to compare the mental behavior status,and the changes of serum Nrf2,MDA,and SOD to assess the oxidative stress level before treatment and after12 weeks,respectively,and to assess the safety of the two groups of patients taking medication.The TCM efficacy of the two groups of patients was judged by the TCM evidence elements score at week 4,week 8 and week 12.Result:1.Experimental study:(1)The effects of Huanglian Jiedu Decoction on the learning and memory ability and anxiety state of AD mice.The open-field experiment showed that the distance and time in the central region were reduced in all groups compared with the control group(P<0.05);the distance and time in the central region were increased in all dosing groups compared with the model group(P<0.05);the comparison between the treatment groups showed that the distance and time in the central region were more in the middle and high dose groups of Huanglian Jiedu Decoction than in the low dose groups of Donepezil and Huanglian Jiedu Decoction(P<0.05).There was no difference between the middle and high dose groups of Huanglian Jiedu Decoction(P>0.05).The new object recognition experiment showed that the cognitive index of mice in each group decreased compared with the control group(P<0.05).Compared with the mice in the model group,the cognitive indices of mice in the medium and high dose Huanglian Jiedu Decoction group and the donepezil group increased(P<0.05);comparison between treatment groups revealed that the cognitive indices of new objects in the low dose Huanglian Jiedu Decoction group decreased compared with the donepezil and medium and high Huanglian Jiedu Decoction groups(P<0.05),and the cognitive indices between the medium and high Huanglian Jiedu Decoction group and the donepezil group were not statistically significant(P>0.05).The results of Morris’ Water Labyrinth Test indicated that the escape latent period of each group was higher than that of the normal group after the 5th Day Orientation Guide(P<0.05);compared with the model group,the escape latency was significantly reduced in all dosing groups(P<0.05);comparison between treatment groups revealed that the escape latency was longer in the HLJDT low-dose group compared with the donepezil and medium-high-dose Huanglian Jiedu Decoction groups(P<0.05),and the difference in escape latency between HLJDT high and medium dose groups and donepezil was not statistically significant(P>0.05).The results of the spatial exploration experiment on day 6 showed that the spatial exploration experiment indexes were reduced in all groups compared with the control group(P<0.05);compared with the model group,the spatial exploration experiment indexes were increased in all dosing groups(P<0.05);comparison between treatment groups showed that the spatial exploration experiment indexes were reduced in the HLJDT low-dose group compared with the donepezil group(P<0.05),while the spatial exploration experiment indexes were reduced in the donepezil The differences between the HLJDT high-dose group and the HLJDT high-dose group were not statistically significant(P>0.05).(2)Effects of Huanglian Jiedu Decoction on the histopathological morphology of the cortex in AD miceThe results of HE staining showed that the cortical areas of the six groups of mice had disorganized cell arrangement and some neural cells had vacuolated degeneration or solidified necrosis compared with the control group.Compared with the model group,the morphology of the cortical area in each group was improved,with clearer tissue structure and fewer degenerated or necrotic cells.Compared with the control group,the neurons in cortex of rats were decreased obviously(P<0.05).compared with the model group,the number of neurons in the cortical area of mice in each dosing group was increased(P<0.05);comparison between treatment groups revealed that the number of neurons in the cortical area of mice in HLJDT medium-high dose and donepezil groups were increased compared with HLJDT low dose group(P<0.05),while HLJDT medium-high dose and There was no difference between HLJDT medium-high dose and donepezil groups.The results of Nissler staining showed that the cortices of the control mice were rich in Nissler bodies(dark blue)and the nuclei were light blue.In the model group,the cortical area was blurred or disappeared,and the nuclei of the cells were fixed and shrunken.The morphology of the cortical area was improved in each drug group compared with that of the model group,with clearer structure of nisomes and fewer cells with nuclei fixation.Compared with the control group,the number of niche in the cortical area of mice in each group was significantly reduced(P<0.05);compared with the model group,the number of niche in the cortical area of mice in the donepezil group,HLJDT low,medium and high dose groups was significantly increased(P<0.05);comparing between treatment groups,the number of niche in the cortical area of mice in HLJDT medium and high dose and donepezil groups was increased compared with HLJDT low dose group(P<0.05),and there was no difference between HLJDT medium-high dose and donepezil groups(P>0.05).(3)Effects of Huanglian Jiedu Decoction on the expression of Aβ1-42 in hippocampal tissues of AD miceCompared with the control group,the number of Aβ1-42 expression in the hippocampus increased in all groups(P<0.05);Aβ1-42 was reduced in all therapy groups than in the model group(P<0.05).;comparison between treatment groups revealed that the number of Aβ1-42 expression was lower in the HLJDT high and medium dose groups compared with the donepezil and HLJDT low dose groups(P<0.05),and there were no significant differences in HLJDT medium versus high-dose(P>0.05).(4)Effects of Huanglian Jiedu Decoction on oxidative stress injury in hippocampus of AD model miceRegarding MDA expression levels in the hippocampus of mice,the level of MDA was higher than that of control group(P<0.05);MDA decreased in all therapy groups than in the model group(P<0.05);comparison between treatment groups revealed that there was no difference in MDA expression between the low-dose groups of donepezil and HLJDT(P<0.05),and MDA decreased with increasing dose in the HLJDT group(P>0.05).In terms of SOD expression levels in the hippocampus of mice,SOD was lower in other groups than in the control group(P<0.05),while SOD was higher than that of the experimental group(P<0.05).;comparison between treatment groups revealed that there was no difference in SOD expression between the low-dose groups of donepezil and HLJDT(P<0.05),and SOD increased significantly with increasing dose in the HLJDT group(P>0.05).In terms of GSH expression levels in the hippocampus of mice,the GSH of other groups was reduced than that of the control(P<0.05);GSH was higher in all therapy groups than in the model group(P<0.05);comparison between treatment groups revealed no difference in GSH expression between the low-dose groups of donepezil and HLJDT(P<0.05),and GSH was increased with increasing dose in the HLJDT group(P>0.05).In terms of ROS expression levels in the hippocampus of mice,ROS increased in the remaining groups compared with the control group(P<0.05);compared with the model group,ROS decreased in all treatment groups(P<0.05);comparison between treatment groups revealed that there was no difference in ROS expression between the donepezil and HLJDT low-dose groups(P<0.05),and ROS decreased in the HLJDT group with increasing dose(P>0.05).(5)Effect of Huanglian Jiedu Decoction on Kea1/Nrf2 signaling pathway in hippocampus of AD model miceImmunofluorescence staining of Nrf2 showed that most of the Nrf2 in the control and model groups was in the cytoplasm and did not transfer to the nucleus,and Nrf2 in the cytoplasm of the model group was significantly reduced,and Nrf2 in the hippocampus of mice brains in the Huanglian Jiedu Decoction as well as Donepezil groups was activated and transferred to the nucleus.In terms of Nrf2 expression levels in the hippocampal nucleus of mice,Nrf2 was elevated in the remaining groups compared with the control group(P<0.05);compared with the model group,Nrf2 was elevated in all treatment groups(P<0.05);comparison between treatment groups revealed that there was no significant difference in Nrf2 expression between donepezil and HLJDT low-dose groups(P<0.05),and HLJDT group with the increase of dose Nrf2 was significantly higher in the HLJDT group with increasing dose(P>0.05).Regarding the expression levels of H0-1 and NQO1 in the hippocampus of mice,compared with the control group,the expression of HO-1 and NQO1 protein decreased in the hippocampus of the remaining groups(P<0.05);compared with the model group,the donepezil group,each Huanglian Jiedu Decoction group was able to increase the expression of HO-1 and NQO1 protein(P<0.05);comparison between treatment groups revealed that the expression of HO-1 and NQO1 in the low dose group of HLJDT and donepezil.were no difference(P<0.05),and the HLJDT group significantly increased HO-1 and NQO1 with the increase of dose(P>0.05).(6)Effects of Huanglian Jiedu Decoction on liver and kidney functions of AD miceThere was no statistical difference in serum ALT,AST,Cr and BUN levels between the groups compared with those before treatment(P>0.05).2.Clinical study:(1)Completion of case recruitmentA total of 60 patients with mild to moderate AD were included in the study.14 cases were disengaged,8 in the control group and 6 in the observation group,due to the impact of the new crown epidemic and other factors during the study,and finally,46 cases finally completed the 3-month trial,with an overall disengagement rate of 23.3%.(2)Baseline data comparisonThere was no statistical difference between the control group and the observation group in terms of pre-treatment sexual general information status,dementia severity,MMSE scale score,ADL scale score,NPI scale score,TCM evidence elements score,serum Nrf2,SOD,and MDA levels(P>0.05).(3)Post-treatment efficacy evaluationWhen comparing the MMSE scale scores,the MMSE scores increased in all groups after 12 weeks of treatment compared to the pre-treatment period(P<0.05).MMSE scores increased in the observation group after 12 weeks of treatment compared with the control group(P<0.05).When comparing the ADL scale scores,the ADL scores decreased in all groups after 12 weeks of treatment compared with those before treatment(P<0.05),and the difference between the two groups was statistically significant(P<0.05).When comparing the NPI scale scores,the NPI scores decreased in all groups after 12 weeks of treatment compared with the pre-treatment period(P<0.05),and in the observation group compared with the control group after 12 weeks of treatment(P<0.05).When comparing the scores of Chinese medical evidence elements,from the 4th week of treatment,the Yang hyperactivity and toxicity evidence in the observation group decreased compared with the pre-treatment and control groups (P<0.05);from the 8th week of treatment,the phlegm turbidity evidence in the observation group decreased compared with the pre-treatment and control groups(P<0.05);at the 12 th week of treatment,the blood stasis evidence in the observation group decreased compared with the pre-treatment and control groups(P<0.05),while the In the 12 th week of treatment,the blood stasis evidence in the observation group decreased compared with that before treatment and compared with that in the control group(P<0.05),while the differences between the other evidence scores in the observation group and before treatment and compared with that in the control group were not statistically significant(P>0.05).The difference of Nrf2、SOD、MDA was significantly higher than that of pre-therapy in 12 weeks.The results showed that there was statistical significance between Nrf2、SOD、MDA(P<0.05).(4)Safety observationThe safety-related laboratory indexes(blood routine,liver and kidney function,four coagulation items,urinary routine,stool routine,and electrocardiogram)were within the normal range during the treatment period in both groups.Two cases of loose stools in the observation group and one case of nausea in the control group were mild,and the dose was reduced in the observation group,while the symptoms disappeared in the control group after no special treatment.There was no statistical significance in the occurrence rate of ADR(P>0.05).Conclusion:1.Experimental research shows that:The intervention of Huanglian Jiedu Decoction effectively improved the learning and memory ability and anxiety status of3 x Tg-AD mice,improved the pathological morphology of cortical areas and protected neurons,decreased the expression level of total Aβ1-42 in hippocampus,improved oxidative stress in hippocampal tissues,activated the Kea1/Nrf2 signaling pathway in hippocampus,and upregulated HO-1 and NQO1 proteins,and did not affect the liver and kidney function of mice.We conclude that Huanglian Jiedu Decoction may activate the Kea1/Nrf2 signaling pathway to upregulate HO-1 and NQO1 proteins,inhibit MDA and ROS,and upregulate SOD and GSH expression to protect neurons and nisomes and improve learning and memory and anxiety status in 3x Tg-AD mice.The low,medium and high doses of Huanglian Jiedu Decoction differed in different index results,but overall,the medium and high doses were superior to the low doses,and none of the three doses produced hepatic or renal toxicity to mice during gavage.2.Clinical research shows that:The combination of Donepezil and Huanglian Jiedu Decoction improved the cognitive function,self-care ability,psycho-behavioral symptoms,some TCM symptoms,and various parameters such as oxidative stress in AD patients,and it was more effective than Donepezil alone,indicating that the addition of Huanglian Jiedu Decoction is more effective in improving the patients’ Western symptoms,TCM symptoms,and also acts as an antioxidant,with better efficacy and high safety. |
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