Dynamic Changes Of Cardiac Sympathetic Nerve Remodeling In Different Periods Of Chronic Heart Failure And The Intervention Effects Of Guizhi Decoction

Objective:1.Clinical observation:To observe the changes of heart rate variability（HRV）,nerve growth factor（NGF）and leukemia inhibitory factor（LIF）in different TCM syndromes of patients with chronic heart failure,and to explore the relationship between TCM syndromes and autonomic neuropathy,as well as the relationship between NGF,LIF and HRV.2.Experimental research:To observe the NGF and LIF expression and abnormal anatomical and functional innervation of cardiac sympathetic nerves in different stages of salt-sensitive hypertension-induced chronic heart failure rats.To prove that the expression of NGF and LIF is an important molecular mechanism affecting anatomical and functional innervation of sympathetic nerves.To observe the effect of Guizhi Decoction on the expression of NGF and LIF in decompensated heart failure and prove that Guizhi Decoction could improve sympathetic nerves remodeling in heart failure by regulating NGF and LIF expression.Methods:1.Clinical observation:Reference《Expert Consensus on Diagnosis and Treatment of Chronic Heart Failure by Integrated Traditional Chinese and Western Medicine》and《Guiding Principles for Clinical Research of New Chinese Medicines》,Traditional Chinese Medicine syndrome differentiation of chronic heart failure is 50patients with yin deficiency syndrome,53 patients with yang deficiency syndrome and 46healthy subjects were included.To observe the changes of HRV,NGF,LIF,left ventricular ejection fraction（LVEF）,N-terminal pro-B type natriuretic peptide（NT-pro BNP）,classification of cardiac function（NYHA）and Lee’s heart failure score in each group.2.Experimental research:（1）Six-week-old DS male rats and SS-13^BNmale rats each twenty-seven were fed with high salt diet（8%Na Cl）.6,9 and 12 weeks after the start of high salt diet feeding were selected as the period of myocardial hypertrophy,compensation and decompensation in the development of heart failure.At each time point,9 rats were selected separately to detect the relevant indicators and another three DS rats and three SS-13^BNrats were used to detect heart rate variability（HRV）.Echocardiography and NT-pro BNP were measured to prove successful induction of heart failure.The changes of heart rate and P-R interval were detected by electrocardiogram.HE and Masson staining were used to observe myocardial morphological changes and collagen deposition in rats.HRV was used to reflect the ability of autonomic nervous regulation.Norepinephrine（NA）,tyrosine hydroxylase（TH）in myocardium and NA in plasma were measured to reflect the synthesis,release and reabsorption of norepinephrine in sympathetic nerve terminals.Western blotting was used to detect the expression of growth-related protein 43（GAP43）and polysialylated neural cell adhesion molecule（PSA-NCAM）in myocardium.TH/GAP43 double immunofluorescence were used to localize cardiac sympathetic nerves regeneration.GAP43/PSA-NCAM determined that the new sympathetic nerves were immature nerves distribution,i.e.“regeneration immature”phenomenon.Tyrosine hydroxylase（TH）and choline acetylaminotransferase（Ch AT）（Double immunofluorescence and Western blotting）were detected in myocardium,and the changes of containing cored vesicles（SGV）and containing clear vesicles（SAGV）in the nerves endings of left ventricle and stellate ganglion were observed by transmission electron microscopy（TEM）to reflect the distribution of sympathetic and vagus in heart and stellate ganglion,which proved that the sympathetic“cholinergic transdifferentiation”.Detection of expression of NGF and LIF protein or m RNA in myocardium reflects the dynamic trend of NGF and LIF expression.（2）Six-week-old DS male rats were divided into model group（DS group,9 rats）and Guizhi Decoction group（GS group,9 rats）,another SS-13^BNrats were given as normal control group(SS-13^BNgroup,9 rats).The dosage of Guizhi decoction was 4.0g crude drug/Kg/day in Guizhi Decoction group.DS group and SS-13^BNgroup were given saline（2m L/day）by gavage.Rats in each group were fed with high salt diet（8%Na Cl）.Guizhi Decoction was administered orally at the 10th weekend of the high salt diet.After 4 weeks of continuous intragastric administration,the rats were executed for detection.General index:Body weight,blood pressure,color doppler echocardiography and electrocardiogram.Myocardial histomorphology and collagen changes were detected by HE and Masson staining.The content of NA in myocardium and plasma and the level of NT-pro BNP in plasma were measured by Elisa.The expression of NGF and LIF m RNA/protein in cardiac tissue was detected by RT-PCR/Western blotting.TH,CHAT,GAP43 and PSA-NCAM protein expression in heart was detected by Western blotting.TH/Ch AT was detected by double immunofluorescence,and the changes of containing cored vesicles（SGV）and containing clear vesicles（SAGV）at the end of left ventricle and stellate ganglion nerve were observed by electron microscopy,which proved that the sympathetic“cholinergic transformation”.TH/GAP43 double immunofluorescence localizes sympathetic regeneration.GAP43/PSA-NCAM confirmed the phenomenon of sympathetic“regeneration immaturity”.Results:1.Clinical observation:The levels of HRV（SDNN、RMSSD、LF、HF）in patients with heart failure in Yin deficiency syndrome group and Yang deficiency syndrome group were lower than those in healthy control group（P<0.05 or 0.01）.The SDNN index of autonomic system in Yang deficiency syndrome group was significantly lower than that in Yin deficiency syndrome group（P<0.01）.There was no significant difference in RMSSD and HF indicators of vagal function between the two groups（P>0.05）.The LF index of sympathetic nerves function in Yang deficiency syndrome group was lower than that in Yin deficiency syndrome group（P<0.01）.The LF/HF index of sympathetic-vagal innervation balance in Yin deficiency group was higher than that in healthy control group（P<0.05）,while that in Yang deficiency group was lower than that in healthy control group（P<0.05）and the LF/HF index in Yang-deficiency syndrome group was significantly lower than that in Yin-deficiency syndrome group（P<0.01）.The NGF index of Yin deficiency syndrome group was significantly higher than that of healthy control group（P<0.01）,while that of Yang deficiency syndrome group was lower than that of healthy control group（P<0.05）.The LIF indexes of Yin deficiency syndrome group and Yang deficiency syndrome group were higher than that of healthy control group（P<0.01）,while Yang deficiency syndrome group LIF index was higher than Yin deficiency syndrome group（P<0.01）.Univariate correlation analysis showed that NGF was positively correlated with SDNN and LF（P<0.05 or 0.01）,while LIF was negatively correlated with SDNN and LF（P<0.01）.Multivariate linear regression analysis showed that NGF was positively correlated with SDNN and LF（P<0.05）,while LIF was negatively correlated with SDNN and LF（P<0.01）.In terms of cardiac function,LVEF index in Yang deficiency group was significantly lower than that of Yin deficiency group（P<0.01）,Lee’s total score of heart failure,NT-pro BNP level and NYHA classification of cardiac function were significantly higher than those of Yin deficiency group（P<0.01）.Experimental research:（1）General index:In DS group,the body weight decreased,blood pressure increased,heart rate slowed down,P-R interval prolonged and cardiac function damaged significantly（NT-pro BNP,LVEF）compared with SS-13^BNgroup in high salt diet6-12 weeks.Heart rate variability showed that the LF/HF ratio of sympathetic-vagal balance index in DS group was higher than that in SS-13^BNgroup at the 6-week（P<0.05）,and significantly lower than that in SS-13^BNgroup at 12-week（P<0.01）.Elisa showed that compared with SS-13^BNgroup,the level of NA in myocardial tissue of DS group decreased（P<0.05）,while the level of plasma NA continued to increase（P<0.05）.HE and Masson staining showed that with the progress of heart failure,the volume of cardiac myocytes increased,the interspace of myocardium widened,inflammatory infiltration,ground glass degeneration,myocardial fibrosis disorder,and a large amount of collagen deposition in DS group.RT-PCR/Western blotting showed that compared with SS-13^BNgroup,NGF m RNA and protein expression in DS group increased first and then decreased（P<0.05）,while LIF m RNA and protein expression keep increasing（P<0.05）.The expression of neuromarker protein showed that the expression of TH,GAP43,PSA-NCAM protein in DS group was higher than that in SS-13^BNgroup at the 6-week（P<0.05）.The levels of TH and GAP43 protein in DS group were lower than those in SS-13^BNgroup at 12 weeks（P<0.05）,PSA-NCAM protein level decreased but it was still higher than SS-13^BNgroup（P<0.01）.CHAT protein level decreased first and then increased relatively.There was no statistical significance compared with SS-13^BNgroup（P>0.05）.Double immunofluorescence showed that compared with SS-13^BNgroup,the co-expression of TH⁺/GAP43⁺,GAP43⁺/PSA-NCAM⁺positive nerve fibers in DS group increased first and then decreased（P<0.05）.The distribution of TH⁺positive nerve fibers in TH⁺/CHAT⁺staining increased first and then decreased（P<0.05）.Distribution of CHAT⁺positive nerve fibers first decreased slightly and then relativity increased,but it was lower than SS-13^BNgroup,there was no statistical difference.Transmission electron microscopy of left ventricle and stellate ganglion showed that compared with SS-13^BNgroup,the sympathetic nerve with containing cored vesicles（SGV）in DS group were damaged,a large number of vacuoles appeared,and the relativity of vagus nerve containing clear vesicles（SAGV）increased.The results suggest that during the transformation from hypertrophy to compensation and then to decompensation,the anatomical innervation of sympathetic nerves is transformed from high to low,while its functional innervation is continuously reduced.（2）General index:Compared with DS group,Guizhi Decoction could slow down the weight loss,decrease blood pressure,increase heart rate,shorten P-R interval and improve cardiac function（NT-pro BNP and LVEF）in the decompensated period of heart failure.Heart rate variability showed that the LF/HF ratio of sympathetic-vagal balance index increased in GS group（P<0.05）.Elisa showed that the level of NA in myocardium increased（P<0.05）and the level of plasma NA decreased（P<0.05）in GS group.HE and Masson staining showed that in Guizhi Decoction group,the volume of myocardial cells decreased,the gap between myocardium became smaller,the infiltration of inflammatory cells decreased,the arrangement of myocardial fibers was neat,and the collagen damage decreased significantly.RT-PCR/Western blotting showed that Guizhi Decoction could increase the expression of NGF m RNA and protein（P<0.05）,and decrease the expression of LIF m RNA and protein in heart（P<0.05）.The expression of double immunofluorescence and neuromarker protein showed that:Guizhi Decoction could increase the co-expression of TH⁺/GAP43⁺positive nerve fibers（P<0.05）,increase the distribution of TH⁺positive nerve fibers in TH⁺/CHAT⁺fluorescence,but had no effect on the positive nerve fibers of CHAT⁺.There was no statistical difference in the co-expression level of GAP43⁺/PSA-NCAM⁺positive nerve fibers（P>0.05）.The expression of nerve markers TH and GAP43 were up-regulated,but the expression of CHAT and PSA-NCAM was not affected（P>0.05）.Transmission electron microscopy of left ventricle and stellate ganglion showed that Guizhi decoction could repair the damage of containing cored vesicles（SGV）of sympathetic nerve in GS rats and alleviating injury of sympathetic nerve vesicles.The results suggest that Guizhi Decoction could promote sympathetic regeneration and improve anatomical innervation of sympathetic nerves by up-regulating NGF expression,reduce the level of LIF,inhibit the“cholinergic transdifferentiation”and increase functional innervation of sympathetic nerves in decompensated heart failure.Conclusion:1.Clinical observation:There are corresponding abnormal changes of sympathetic and vagal in different TCM syndromes of chronic heart failure.Syndrome of Yin deficiency is characterized by sympathetic hyperactivity,while syndromes of Yang deficiency are characterized by sympathetic dysfunction.Vagus in both groups were damaged to varying degrees.With the progress of heart failure,the degree of autonomic neuropathy gradually increased.There is a correlation between autonomic nervous index（SDNN,LF）and NGF and LIF levels in patients with chronic heart failure.NGF is positively correlated with autonomic nerve and LIF is negatively correlated with autonomic nerve.It can be concluded that NGF and LIF are important nerve factors in response to cardiac autonomic neuropathy,and regulating NGF and LIF may play a role in improving the autonomic neuropathy of heart failure.2.Experimental research:（1）During the hypertrophic period of heart failure rats induced by salt-sensitive hypertension,increased expression of NGF in the heart leads to increased sympathetic regeneration,which leads to high anatomical innervation of cardiac sympathetic.Meanwhile,up-regulated LIF induces regeneration and immaturation of the new sympathetic,which counteracts the excessive innervation of the sympathetic nerves during myocardial hypertrophy.During the period from myocardial hypertrophy to compensation and then to decompensation,the expression of NGF was down-regulated,the level of sympathetic regeneration was down-regulated,and the sympathetic nerves showed anatomical denervation,while the expression of LIF was continuously increased,which induced the sympathetic“cholinergic transdifferentiation”,leading to functional denervation of sympathetic nerves.（2）Guizhi Decoction could increase the level of sympathetic regeneration and inhibit the“cholinergic transdifferentiation”of sympathetic in decompensated heart failure,improve anatomical and functional denervation of sympathetic nerves,and regulate the balance of sympathetic innervation.The mechanism of action is closely related to the regulation of NGF and LIF expression.