| Obstructive Sleep Apnea Hypopnea Syndrome(OSAHS)is a common clinical Sleep disorder,which is related to the incidence and mortality of various chronic diseases and does great harm to human body.OSAHS is often combined with multi-system complications,especially cardiovascular diseases.In addition,hyperlipidemia and arteriosclerosis,which can further induce cardiovascular diseases,have also been gradually concerned.It is found that OSAHS and its complications often interact with each other,increased the patient’s body and the economic burden.At present,the treatment of OSAHS patients mainly consists of using.ventilator combined with exercise therapy,but there are still a part of patients with poorly controlled,who’s complications control is poorer.OSAHS is treated by traditional Chinese medicine in the holistic treatment of patients with primary disease and complications,and has achieved good curative effect with few side effects,which can effectively improve the symptoms of patients.However,there are still some problems such as unstandardized TCM syndrome differentiation and limited research data of OSAHS.Hyperlipidemia is a condition caused by abnormal lipid metabolism or movement.It can cause systemic atherosclerosis resulting in coronary heart disease.peripheral vascular disease and other arteriosclerosis disease which harm to human health.It often accompanies with OSAHS,which both affect and aggravate each other.Commonly used lipid-lowering drugs have the risk of affecting liver function,causing myositis and myopathy,and the combination of drugs may increase the occurrence of these adverse reactions.Traditional Chinese medicine has a significant therapeutic effect on lipid-lowering.and also has the advantages of coordinating western medicine in lipid-lowering,reducing its clinical dosage and side effects.Chinese medicine considers hyperlipidemia as "turbidness of phlegm" and "blood stasis",while arteriosclerosis and OSAHS also have turbidness of phlegm and blood stasis.Phlegm-stasis syndrome is the common syndrome type.The joint treatment of phlegm and blood stasis is the common treatment.Danlou tablet is one of the representative prescriptions of the joint treatment of phlegm and blood stasis,which can regulate blood lipids,improve arteriosclerosis and treat cardiovascular and cerebrovascular diseases.However.whether it is effective in OSAHS induced hyperlipidemia and arteriosclerosis still needs to be further studiedChronic intermittent hypoxia(CIH).a major pathological factor of OSAHS.can lead to dyslipidemia and arteriosclerosis,thus promoting the occurrence and development of cardiovascular and cerebrovascular diseases.Studies have shown that CIH can lead to dyslipidemia and arteriosclerosis through HIF-1-Angptl4 signaling pathway.Previous studies have confirmed that Danlou tablet has anti-inflammatory and endothelial protective effects,while is suitable for OSAHS-induced dyslipidemia and arteriosclerosis.but the mechanism is still unclear.In this study,Apolipoprotein E-/-mouse arteriosclerosis model was treated with CIH,to observe role of the Hif-1-Angptl4 signaling pathway and its possible upstream target Fih-1 in cih-induced AS.Meanwhile,the intervention of Danlou tablet was given to observe the intervention effect and explore the mechanism of its intervention in intermittent hypoxia induced arteriosclerosis and dyslipidemia.Part 1 Study on Chinese medicine syndrome of obstructive sleep apnea hypopnea syndrome and its complicationsObjective:This study retrospectively analyzed the medical history,complications and TCM syndromes of hospitalized patients with OSAHS,and analyzed the characteristics and distribution of OSAHS and complications as well as their relationship with sleep monitoring results.Methods:A single-center retrospective study was performed and included in the respiratory department of Guang’anmen hospital of the Chinese academy of traditional Chinese medicine from January 2014 to December 2018.There were 77 patients diagnosed as OSAHS with complete data.According to the consensus of experts on TCM diagnosis and treatment of snoring syndrome,it is divided into six syndromes,including internal obstruction of phlegm hygrosis.phlegm and blood stasis,accumulation of phlegm heat.qi deficiency.phlegm and blood stasis,lung and spleen qi deficiency,lung and kidney deficiency.A database was established to analyze the general situation of patients,the rule of TCM syndromes and their relationship with sleep monitoring results,OSAHS complications and their relationship with TCM syndromes.Results:1 General information:A total of 77 OSAHS patients were included in this study,including 40 males and 37 females.The oldest patients were 80 years old,and the youngest patients were 31 years old.The average age was 63.82±11.44 years old,and the average BMI28.91±4.45kg/m2.Among them,there are 28 patients with mild OSAHS,19 patients with moderate OSAHS.and 30 patients with severe OSAHS.BMI of severe OSAHS patients was significantly higher than that of mild and moderate patients,P<0.05.2 TCM syndrome analysis2.1 TCM syndrome distribution:There were 26 patients with phlegm and blood stasis syndrome(33.8%);19 patients with internal obstruction of phlegm hygrosis syndrome(24.7%);15 patients with accumulation of phlegm heat syndrome(19.5%);11 patients with qi deficiency,phlegm and blood stasis syndrome(14.3%).There were 4 patients with spleen and kidney deficiency(5.2%)and 2 patients with lung and spleen qi deficiency(2.6%).2.2 TCM syndromes and syndromes:15 patients had accompanied symptoms(19.5%).Among them,there were 9 cases of phlegm and blood stasis+spleen and kidney deficiency,2 cases of lung and spleen qi deficiency+internal obstruction of phlegm hygrosis,and 4 cases of spleen and kidney deficiency+internal obstruction of phlegm hygrosis.Pure positive evidence included 51 cases of syndrome of phlegm and blood stasis,internal obstruction of phlegm hygrosis,and accumulation of phlegm heat.There were 26 cases of deficiency and empirical mixed syndrome.2.3 Comparison between TCM syndromes and sleep monitoring results2.3.1 Comparison of TCM syndromes AHI in each group:AHI in the two groups of internal obstruction of phlegm hygrosis and accumulation of phlegm heat was significantly smaller than that in the three groups of phlegm and blood stasis.qi deficiency,phlegm and blood stasis,spleen and kidney deficiency,P<0.05,with statistical significance.There was no significant difference of AHI in three groups of phlegm and blood stasis.qi deficiency,phlegm and blood stasis.spleen and kidney deficiency,P>0.05,and no significant difference of AHI in the two groups of internal obstruction of phlegm hygrosis and accumulation of phlegm heat.P>0.05.2.3.2 Comparison of LSpO2 among patients with TCM syndromes in each group:LSpO2 in the two groups of internal obstruction of phlegm hygrosis and accumulation of phlegm heat was significantly higher than that in the three groups of phlegm and blood stasis,qi deficiency,phlegm and blood stasis,spleen and kidney deficiency,P<0.05,with statistical significance.There was no significant difference in LSpO2 between the two groups of obstruction of phlegm hygrosis and accumulation of phlegm heat(P>0.05),and there was no significant difference in LSpO2 between the three groups of phlegm and blood stasis,qi deficiency.phlegm and blood stasis,phlegm and blood stasis+spleen and kidney deficiency,(P>0.05).2.3.3 Comparison of severity of OSAHS in TCM syndromes of each group:mild and moderate OSAHS were dominant in patients with internal obstruction of phlegm hygrosis and accumulation of phlegm heat syndrome,while moderate and severe OSAHS were dominant in patients with phlegm and blood stasis,qi deficiency,phlegm and blood stasis,phlegm and blood stasis+spleen and kidney deficiency.2.4 Complications of TCM syndromesStatistics of patients with OSAHS complications:Hyperlipidemia was the most common complication.accounting for 56 cases(72%),followed by hypertension(52 cases.67%),chronic respiratory disease(32 cases.41%),diabetes(28 cases,36%),coronary heart disease(27 cases,35%),gastroesophageal reflux disease(24 cases,31%),and stroke(10 cases,13%).Among them,7 patients had no complications,6 patients had one complication,15 patients had two complications,19 patients had three complications,and 30 patients had three or more complicationsThe relationship between TCM syndromes and OSAHS complications:OSAHS patients with coronary heart disease often witness internal obstruction of phlegm hygrosis and internal accumulation of phlegm heat.Hypertension is common in internal obstruction of phlegm hygrosis and phlegm and blood stasis syndrome.Stroke is common in the syndrome of phlegm and blood stasis.Diabetes mellitus is common in the syndrome of phlegm and blood stasis.Hyperlipidemia is common in phlegm and blood stasis and internal obstruction of phlegm hygrosis.Gastroesophageal reflux is common in the syndrome of accumulation of phlegm heat.Respiratory diseases are common in internal obstruction of obstruction of phlegm hygrosis.Conclusion:The syndrome distribution of OSAHS in traditional Chinese medicine is phlegm and blood stasis syndrome>internal obstruction of phlegm hygrosis syndrome>accumulation of phlegm heat syndrome>qi deficiency,phlegm and blood stasis syndrome>spleen-kidney deficiency syndrome>lung and spleen qi deficiency syndrome.It is mainly based on excess syndrome,followed by intermingled deficiency and excess syndrome.The most common syndrome is syndrome of phlegm and blood stasis.The sleep monitoring results of different syndromes were different.Mild and moderate OSAHS were dominant in patients with internal obstruction of phlegm hygrosis and accumulation of phlegm heat syndrome.while moderate and severe OSAHS were dominant in patients with phlegm and blood stasis,qi deficiency,phlegm and blood stasis,phlegm and blood stasis+spleen and kidney deficiency.OSAHS is often accompanied by a variety of complications,and hyperlipidemia is the most common one.Other cardiovascular and cerebrovascular diseases,metabolic diseases,digestive diseases and respiratory diseases can be seen.Syndrome of traditional Chinese medicine has certain relationship with the occurrence of complications,which can guide the treatment of OSAHS and its complications.Part 2:Study on intervention of Danlou tablet on chronic intermittent hypoxia induced dyslipidemia and arteriosclerosis in mice modelStudy 1:Evaluation of curative effect on intermittent hypoxia-induced dyslipidemia and arteriosclerosis in mice model treated with Danlou tabletObjective:To evaluate the efficacy of Danlou tablet in the treatment of intermittent hypoxia-induced dyslipidemia and arteriosclerosis in mice.Methods:16 male ApoE-/-mice aged 12 weeks were divided into 4 groups,4 in each group,and no CIH modeling was performed in Sham group.CIH+normal saline group was treated with CIH,and normal saline(2ml/time)was given to the stomach once a day.CIH+Angptl4 antibody group was treated with CIH,and Angptl4 immunohistochemical antibody(30mg/kg)was injected intraperitoneally every week.The CIH+Danlou tablet group was treated with CIH,and Danlou tablet(250mg/kg)were given by gavage once a day.Samples were collected after 4 weeks of intervention in each group,blood was collected after orbit,and serum was centrifuged.The animals were killed,the epididymal fat pad,and the heart tissue samples was taken storeing in the-80℃ refrigerator.After the animals were killed,phosphate buffer was slowly irrigated from the left ventricle to separate the aortic arch.Serum lipid was detected by ELISA.Serum lipoprotein was detected by kit.Serum LPL activity was detected by kit.Morphological properties of arterial plaques were observed by HE and immunohistochemistryResults:1 Serum lipids and lipoproteins of ApoE-/-mice in each group were determinedTC,TG,LDL and VLDL in CIH group were compared with those in Sham group.P<0.01.The difference between the two groups was significant and statistically significant,indicating that CIH could lead to dyslipidemia in mice.The results of TC,TG,LDL and VLDL in the Angptl4 antibody group were compared with those in the CIH group,with P<0.01.The difference between the two groups was significant and statistically significant.The Angptl4 antibody could alleviate the dyslipidemia induced by CIH and reduce the blood lipid.TC,TG,LDL and VLDL of Danlou tablet group were compared with that of CIH group,P<0.01,the difference between the two groups was significant,with statistical significance.Danlou tablet can intervene the dyslipidemia induced by CIH,improve the blood lipid metabolism.2 Serum lipoprotein lipase(LPL)activity in ApoE-/-mice was measuredLPL activity in CIH group was significantly lower than that in Sham group,P<0.01.and the difference between the two groups was statistically significant.Compared with Sham group,LPL activity in serum of mice in CIH group was significantly decreased,indicating that CIH could reduce LPL activity,hinder the clearance of blood lipids,and cause the increase of blood lipids.The LPL activity of the Angptl4 antibody group was significantly different from that of the CIH group(P<0.01),suggesting that the Angptl4 antibody group may increase the LPL activity by neutralizing Angptl4,thereby reducing blood lipid.Compared with CIH,the LPL activity of Danlou tablet group was P<0.01,and the difference between the two groups was significant,with statistical significance.The LPL activity of Danlou tablet was significantly increased after the intervention,and the lipid metabolism of blood was improved by increasing the LPL activity of Danlou tablet.3 Observation on the morphology and property of arterial plaqueCompared with Sham group,the area of atherosclerotic plaques,arterial wall thickening and lumen narrowing in CIH group were significantly increased.Compared with the CIH group,the area of atherosclerotic plaque in the Angptl4 group and the Danlou tablet group was significantly reduced,the thickness of the arterial wall was narrowed to a certain extent,and the pathological symptoms of atherosclerosis were alleviated.Compared with the CIH group,the CD68 staining area of atherosclerotic plaques in the Angpt14 antibody group and the Danlou tablet group was significantly reduced,the staining intensity was weakened,the thickness of the arterial wall was narrowed to a certain extent,and the pathological symptoms of atherosclerosis were alleviated.Conclusion:Intermittent hypoxic treatment can aggravate atherosclerotic lesions and increase blood lipids in ApoE-/-mice.By the treatment of Angpt14 antibody and Danlou tablet,the atherosclerosis of mice was alleviated to a certain extent.and the blood lipids decreased to different degrees.This experiment verified the effectiveness of the intervention of Danlou tablet in the treatment of cih-induced dyslipidemia and arteriosclerosis,and the intervention mechanism still needs to be further explored.Study 2:Study on the mechanism of intervention of Danlou tablet on dvslipidemia and arteriosclerosis induced by chronic intermittent hypoxiaObjective:To explore the Hif-1-Angptl4 pathway and its possible upstream target Fih-1 in the process of chronic intermittent hypoxia-induced dyslipidemia and arteriosclerosis,as well as the possible intervention mechanism of Danlou tablet.Methods:Samples of epididymal fat pad and heart tissue from each group were taken and divided into 4 groups according to experiment 1.Qrt-PCR was conducted to detect the mRNA expressions of Fih-1,Angptl4,Hif-1 and LPL in epididymal fat pad and heart tissue of each group of mice.The protein levels of Fih-1,Angptl4,Hif-1 and LPL in epididymal fat pad and heart tissues were detected by Western blot.Results:1 Qrt-pcr results of epididymal adipose cushion tissues in ApoE-/-miceCompared with the Sham group,the CIH group of FIH-1 mRNA expression level decreased significantly,Angptl4 mRNA expression level increased significantly.HIF-1 mRNA expression level increased significantly,P<0.01,the difference had obvious statistical significance;LPL mRNA expression level showed no significant difference.P>0.05,the difference was not statistically significant.Compared with CIH group,the expression level of FIH-1 mRNA.LPL mRNA.and Angptl4 mRNA in the Angptl4 antibody group were significantly increased,while the expression level of HIF-1 mRNA was decreased.The difference was statistically significant(P<0.01).Compared with CIH group,the expression level of FIH-1 mRNA and LPL mRNA in the Danlou tablet group were significantly increased,while the expression levels of Angptl4 mRNA and HIF-1 mRNA in Danlou tablet group were significantly decreased.P<0.01,and the difference was statistically significant.2 Results of Qrt-pcr detection in heart tissue of ApoE-/-miceCompared with Sham group,after CIH intervention.the expression level of FIH-1 mRNA was significantly decreased,and the expression level of HIF-1 mRNA was significantly increased,P<0.01,with statistically significant differences.There was no significant difference in the expression levels of Angptl4 mRNA and LPL mRNA.Compared with the CIH group,the expression levels of FIH-1 mRNA in the Angpt14 antibody group and the Danlou tablet group were significantly increased,and the expression levels of HIF-1 mRNA were significantly decreased,P<0.01,with statistically significant differences.There was no significant difference in the expression levels of Angptl4 mRNA and LPL mRNA.3 Western blot analysis of epididymal adipose pad tissue from ApoE-/-miceCompared with Sham group,the expression level of FIH-1 protein in CIH group was significantly decreased,while the expression levels of Angptl4 and HIF-1 protein were significantly increased,P<0.01,with statistically significant differences.There was no significant difference in the expression level of LPL protein.P>0.05.Compared with the CIH aroup,the expression levels of FIH-1 and LPL proteins in the Angptl4 group and the Danlou tablet group were significantly increased,and the expression levels of Angptl4 and HIF-1 proteins were significantly decreased.P<0.01,with statistically significant differences.4 Western blot analysis of heart tissue from ApoE-/-mice.Compared with Sham group,the expression level of FIH-1 protein was significantly decreased after CIH intervention,and the expression level of HIF-1 protein was significantly increased,P<0.01,with statistically significant difference.There was no significant difference in the expression level of Angptl4 and LPL protein,P>0.05,and the difference was not statistically significant.Compared with the CIH group,the expression level of FIH-1 protein in the Angptl4 antibody group and the Danlou tablet group was significantly increased,and the expression level of HIF-1 protein was significantly decreased,P<0.01,and the difference was statistically significant.There was no significant difference in the expression level of Angptl4 and LPL protein,P>0.05,and the difference was not statistically significant.Conclusion:Danlou tablet may affect the Hif-1-Angptl4 signaling pathway of epididymal fat pad and the possible upstream target of this pathway Fih-1 for the treatment of chronic intermittent hypoxia induced dyslipidemia and arteriosclerosis,which provides a research basis for the intervention of traditional Chinese medicine treating phlegm and blood stasis treatment with OSAHS-induced dyslipidemia and arteriosclerosis. |
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