Mechanism of Chinese Medicine Herbs Effects on Chronic Heart Failure Based on Metabolic Profiling

Herbs and Helpers

ORIGINAL: https://doi.org/10.3389/fphar.2017.00864

• 1School of Preclinical Medicine, Beijing University of Chinese Medicine, Beijing, China

• 2Beijing University of Chinese Medicine, Dongfang Hospital, Beijing, China

• 3FengNing Chinese Medicine Hospital, FengNing, China

Chronic heart failure (CHF) is a major public health problem in huge population worldwide. The detailed understanding of CHF mechanism is still limited. Zheng (syndrome) is the criterion of diagnosis and therapeutic in Traditional Chinese Medicine (TCM). Syndrome prediction may be a better approach for understanding of CHF mechanism basis and its treatment. The authors studied disturbed metabolic biomarkers to construct a predicting mode to assess the diagnostic value of different syndrome of CHF and explore the Chinese herbal medicine (CHM) efficacy on CHF patients. A cohort of 110 patients from 11 independent centers was studied and all patients were divided into 3 groups according to TCM syndrome differentiation: group of Qi deficiency syndrome, group of Qi deficiency and Blood stasis syndrome, and group of Qi deficiency and Blood stasis and Water retention syndrome. Plasma metabolomic profiles were determined by UPLC-TOF/MS and analyzed by multivariate statistics. About 6 representative metabolites were highly possible to be associated with CHF, 4, 7, and 5 metabolites with Qi deficiency syndrome, Qi deficiency and Blood stasis syndrome, and Qi deficiency and Blood stasis and Water retention syndrome (VIP > 1, p < 0.05). The diagnostic model was further constructed based on the metabolites to diagnose other CHF patients with satisfying sensitivity and specificity (sensitivity and specificity are 97.1 and 80.6% for CHF group vs. NH group; 97.1 and 80.0% for QD group vs. NH group; 97.1 and 79.5% for QB group vs. NH group; 97.1 and 88.9% for QBW group vs. NH group), validating the robustness of plasma metabolic profiling to diagnostic strategy. By comparison of the metabolic profiles, 9 biomarkers, 2-arachidonoylglycerophosphocholine, LysoPE 16:0, PS 21:0, LysoPE 20:4, LysoPE 18:0, linoleic acid, LysoPE 18:2, 4-hydroxybenzenesulfonic acid, and LysoPE 22:6, may be especially for the effect of CHM granules. A predicting model was attempted to construct and predict patient based on the related symptoms of CHF and the potential biomarkers regulated by CHM were explored.

This trial was registered with NCT01939236 (https://clinicaltrials.gov/).

Introduction

Chronic heart failure (CHF), as a complex clinical syndrome, results from any structural or functional cardiac disorder that impairs the ability of the ventricle to fill with or eject blood (Hunt et al., 2005). In 2000, an international cooperation research program on cardiovascular disease in Asia (InterASIA) was made, which included 15,518 urban or rural residents from 35 to 74 years old. The results showed that the prevalence of heart failure was 0.9% for the general population, 0.7% for the males, and 1.0% for the females, indicating that there were ~4 million heart failure targets in China (Gu et al., 2003). CHF is the only cardiovascular disease with an increasing hospitalization burden and an ongoing drain on health care expenditures, for its complex molecular mechanisms cannot be easily deciphered (Ramani et al., 2010). As a complementary alternative, Traditional Chinese Medicine (TCM) may be to improve the state of CHF. Zheng (syndrome) is the key pathological principle of TCM. All diagnostic and therapeutic methods in TCM are based on the differentiations of syndrome, and this concept has been used for thousands of years in China (Gu, 1956; Li et al., 2007). Syndrome differentiation was based on information from traditional four diagnostic methods. For the complexity and multilevel relationships of four diagnostic methods, a mode which could distinguish syndrome differentiation and be verified is imperative. Many techniques of data mining are applied to syndromes in TCM (Lukman et al., 2007; Shi and Zhou, 2007; Gao et al., 2010; Yao et al., 2011). Chen et al. proposed a discovery algorithm based on revised mutual information to discover syndromes for chronic renal failure (Chen et al., 2007). In regards to coronary artery disease, Liu et al. designed standardization scale on inquiry diagnosis and constructed this diagnostic model by using the method of multi-label learning (Liu et al., 2010). With many achievements have been made in syndrome differentiation, there are still some problems left, deserving further discussion (Wang et al., 2009; Lu et al., 2012). Syndromes-identified techniques such as multi-label learning could identify syndrome information in TCM more effectively, and solve the multi-label problems of one sample with several syndromes. While there are many research challenges of multi-label learning to be addressed in the future, such as multi-label data usually suffers from inherent class imbalance and unequal misclassification costs. Metabolomics is distinctive for its overall concept and dynamic character, which may be an effective tool to reveal the scientific connotation of TCM. Metabolic alterations are measured in response to disease progression (Cheng et al., 2015). LC-MS based methods provide more compatible technique and high quality data for sensitive detection of small-molecule metabolites with robust reliability and reproducibility (Gika et al., 2007). Abnormal metabolism may characterize syndrome differentiation. Our previous metabolomics study on blood stasis syndrome of CHF showed glucose metabolism and lipid metabolism disorders reinforce each other, which results a deterioration of coronary artery disease with blood stasis syndrome. These metabolites maybe used as indicators of clinical diagnosis and treatment of coronary artery disease (Wang et al., 2011). Wang et al. concluded that NMR-based metabolomics approach demonstrated alteration of energy metabolism and other potential biological mechanisms underlying CHF (Wang et al., 2013b). In this paper, we try to discover comprehensive metabolomic characteristics of CHF and its syndrome differentiation for accurate clinical diagnosis. A syndrome predictive method for CHF was to build with non-targeted metabolomics and potential biomarkers related to CHF syndrome differentiation were to explore. The predictive performance would be validated and these potential biomarkers would be used as predictors to diagnose the patients. Besides, special metabolic characteristic of CMH on CHF would also be elaborated.

Materials and Methods

Patients and Study Design

The study included 110 patients with chronic heart failure from 11 hospitals (The Second Affiliated Hospital of Beijing University of Chinese Medicine, Zhengzhou Hospital of TCM in Henan Province, The Affiliated Hospital to Changchun University of CM, Guang’anmen Hospital which was Affiliated to China Academy of Chinese Medicine Sciences, Hubei Provincial Hospital of TCM, Wuhan Hospital of TCM, Hospital of T.C.M.S Shijingshan District, Beijing Changping Hospital, Hospital of T.C.M.S Beijing Miyun County, Beijing Changping Hospital of Integrated Chinese and Western Medicine, TCM Hospital of Beijing Huairou and TCM Hospital of Tongzhou District) in China between May 2010 and December 2014. During the same period, 54 normal healthy participants of matched age were included from medical center of Dongzhimen Hospital as controls.

Diagnostic Criteria

Diagnostic criteria of CHF: 2007 China Guideline for the Diagnosis and Treatment of CHF (Chinese Society of Cardiology of Chinese Medical Association and Editorial Board of Chinese Journal of Cardiology, 2007). Heart function standard: The Criteria for Diagnosis and Treatment of Heart Disease first published by the New York Heart Association (NYHA) (Feinstein, 1964). Syndrome differentiation of CHF patients followed the TCM differentiation standard: according to the Guiding Principles for the Clinical Study of New Drugs in Traditional Chinese Medicine released in 2002 (Zheng, 2002).

Inclusion Criteria

Primary heart disease in this research was Coronary Heart Disease (CHD), which could be diagnosed by coronary computed tomography, coronary angiography, limb-salvage Q wave for electrocardiogram (ECG), history of acute myocardial infarction, ECG test, radionuclide examination support, etc. The included patients also had no history of taking antihypertensive drugs and exhibited a blood pressure under 160/100 mmHg or of hypertension. Following symptoms and signs were observed with a history of CHD: fatigue, difficulty breathing, fluid retention (edema), left ventricular enlargement, NYHA functional classification II or III, and end systolic volume of left ventricular increase and left ventricular ejection fraction (LVEF)<40. All subjects between 40 and 75 years old; If there was a “No” answer for any issue, the subject could not enter into this research.

Exclusion Criteria

Patients with one of the following diseases were excluded: (1) serious valvular heart disease; (2) cardiomyopathy; (3) pericardial disease; (4) congenital heart disease; (5) cardiac shock; (6) acute myocardial infarction (AMI) within 4 weeks; (7) acute myocarditis or serious arrhythmia with variation in hemodynamics. Patients who suffer from pulmonary artery hypertension caused by cor pulmonale, pulmonary embolism, or stroke within a half year were also excluded. Patients who suffer from serious hepatic or renal deficiency. Patients who suffer from diseases of blood system or malignant tumor. Patients who suffer from diabetes mellitus with serious complications, hyperthyrea, or hypothyrea. Patients who suffer from infection. Pregnant women or women in lactation. Patients with mental disorders. Patients with infectious disease or who join in other trials within 2 months of the present study.

All qualified participants had signed informed consent prior to the enrollment. This study was conducted according to the principles of the Declaration of Helsinki and the principles of Good Clinical Practice.

All patients formed the chronic heart failure group, and were further divided into 3 groups according to TCM syndrome differentiation, including group of Qi deficiency syndrome (QD), group of Qi deficiency and Blood stasis syndrome (QB), and group of Qi deficiency and Blood stasis and Water retention syndrome (QBW). The normal healthy participants composed the NH group.

Besides, according to previous research (Wang et al., 2013a), the most syndrome of CHF was Qi deficiency and Blood stasis syndrome, so 64 of the participants with CHF of Qi deficiency and Blood stasis syndrome (QB group) were enrolled in a randomized double-blind controlled clinical trial. According to the 2007 China Guidelines for the Diagnosis and Treatment of Chronic Heart Failure (Chinese Society of Cardiology of Chinese Medical Association and Editorial Board of Chinese Journal of Cardiology, 2007), all patients in the two groups received the standardized western medicine treatment, which includes angiotensin-converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARBs), b-blockers, and diuretics. Except the standardized western medicine treatment, subjects with the CHM treatment were defined as the CHM group, and subjects with the placebo intervention were defined as the placebo group. The subjects were treated with CHM granules, made from Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao (huangqi, 60 g), Codonopsis pilosula (Franch.) Nannf (dangshen, 15 g), Salvia miltiorrhiza Bge (danshen 15 g), Paeonia lactiflora Pall (chishao, 15 g), Prunus davidiana (Carr.) Franch. (taoren, 10 g), and Carthamus tinctorius L. (honghua, 10 g) or placebo granules twice per day for 4 weeks. The CHM granules and placebo granules were offered by Beijing Kang Rentang pharmaceutical industry (Beijing, China). Finally, 31 patients were in CHM group, and 33 were in placebo group.

Plasma samples were collected on morning of the first and the twenty-eighth day after enrollment and were immediately frozen at −80°C. Six minutes of walking test (6 MWT) and echocardiography were also detected at the same time. Both the observer of the 6 MWT and the echo technician were blind to the allocation of the patients.

After several preliminary experiments by different mixtures of methanol, acetonitrile, and methanol/acetonitrile (1:1), finally the extraction solvent was acetonitrile. By mixing equal amounts of plasma (30 μL) from all samples, pooled quality control samples were prepared to ensure data quality for metabolic profiling. Detailed sample methods were shown in the Supplementary Material.

This research was approved by the Institutional Committee on Human Research of Beijing University of Chinese Medicine (No. 200807007), and this trial is registered with NCT01939236 (Luo et al., 2015).

Ultra Performance Liquid Chromatography/Time of Flight Mass Spectrometry (UPLC-TOF MS) Metabolomics Study Analysis

The liquid chromatographic separation of processed plasma was conducted on a 100*2.1-mm ACQUITY UPLC ° R BEH C18 1.7-um column (Lot No. 0252350221) using an ACQUITY Ultra Performance LC (Waters Corporation, Milford Massachusetts, USA), whereas mass spectrometry was performed on a SYNAPT G2 Quadrupole-Time of Flight system (Waters Corporation, Milford Massachusetts, USA). With a random-number generator in Excel (Microsoft, Redmond, Washington), 3 sequences for samples in discovery phase, validation phase and placebo group were assigned by the study administrator. During analyses of the sample sequence, after each 20 injections, then 1 quality control sample was run to check the stability of system. The acquired mass spectrometry data were exported to data format (.centroid) files by Masslynx Software (version 4.0, Waters Corporation). Data pre-treatment procedures, such as nonlinear retention time alignment, peak discrimination, filtering, alignment, matching, and identification, were performed in Progenesis QI (34 Maple Street, Milford Massachusetts, 01757, USA), and retention time and the mass-to ratio data pairs as the parameters for each ion. The UPLC-QTOF-MS characteristic chromatogram of CHM granules (6 herbs) was identified, and detailed method was shown in the Supplementary Material. Disturbed metabolites and metabolic pathways were identified by open database sources, including Human Metabolome Database (http://www.hmdb.ca/), METLIN (https://metlin.scripps.edu/), KEGG (http://www.genome.jp/kegg/pathway.html), and MetaboAnalyst (http://www.metaboanalyst.ca/faces/home.xhtml)…

Source: Frontiers

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The sugar industry blocked research linking sucrose to heart disease and cancer from publication 50 YEARS ago, damning report reveals

Herbs and Helpers

The researchers at the University of California at San Francisco have uncovered data showing the sugar industry hid research linking sugar to cancer in 1968

New documents show the Sugar Association funded an animal experiment called Project 259 to evaluate sucrose’s effects on cardiovascular health
But when the data showed a clear link between sucrose and poor heart health, they pulled the plug

The researchers say that, had this paper been published in 1968, it would have led to scrutiny and even regulation of sugar by the FDA

The sugar industry blocked the release of a study showing sucrose directly increases the risk of heart disease and cancer in 1968, newly-uncovered documents reveal.

The research, which was funded and designed by the sugar industry, was intended to dispel fears that fructose-containing sugars affect blood lipids.

But internal correspondence uncovered by researchers at the University of California at San Francisco, show that industry leaders pulled the plug on its publication after getting wind that it would prove the clearest link between sugar and disease ever found.

The finding, published today in PLOS Biology, is the latest in a series of bombshell reports from investigative researcher Dr Cristin Kearns and co-author Dr Stanton Glantz, who was the first researcher to reveal Big Tobacco was hiding research on the danger of cigarettes in 1996.

Last year the duo sent shockwaves through the nutrition world with a study that showed the sugar industry had paid Harvard University’s most respected nutrition scientist to play down the health dangers of sugar, and demonize fats.

Speaking to Daily Mail Online, they say that, had this new study been published in 1968 as planned, it would have automatically triggered a review of sucrose by the US Food and Drug Administration, which would have likely led to regulation of sugar.

Instead, they say, it has taken five decades for the scientific community to reach relative agreement that sugar is bad for you, and has a direct link to cancer and heart disease.

‘The sugar industry has been playing the same games as Big Tobacco to protect their financial interests,’ Dr Glantz told Daily Mail Online.

‘The more we look, the more we see that the sugar industry has had a sophisticated understanding of science for decades, sophisticated enough to manipulate it.

‘This study, if it had been published, would have been quite cutting edge for its time. Had that work moved forward, it would’ve advanced the triglycerides-sugar debate forward by decades.

‘That’s why they killed it.’

Today’s paper – the third collaboration between Glantz and Cearns, and the fourth on this subject for Cearns, a dentist-turned-investigator – is based on a review of archived industry documents.

It reveals the Sugar Research Foundation (SRF), now known as the Sugar Association, funded an animal experiment called Project 259 to evaluate sucrose’s effects on cardiovascular health.

The sugar industry has had a sophisticated understanding of science for decades, sophisticated enough to manipulate it Co-author Dr Stanton Glantz

But, as with many research papers, it got delayed. The researcher, excited by his findings, went to the SRF asking for another boost in funding to take his work further.

Reviewing the data, which indicated an association with heart disease and bladder cancer, the SRF pulled the plug on the entire project.

Dr Cearns explains that this study would have triggered scrutiny or regulation of sucrose.

At the time, the FDA was operating under something called the Delaney clause, which made is compulsory to scrutinize or regulate anything that was found to have a cancerous link based on an animal study.

In fact, a year before, the SRF had criticized animal studies. For unknown reasons, the foundation went on to fund its own animal study – but shut it down when they heard the damning findings.

Even today, the Sugar Association denies that sugar has any direct detrimental health impacts beyond weight gain.

Last year the Sugar Association criticized a mouse study suggesting a link between sugar and the growth and spread of tumors.

They said ‘no credible link between ingested sugars and cancer has been established.’

The analysis of the industry’s own documents, in contrast, suggests the industry knew of animal research indicating this link, and halted funding to protect its commercial interests half a century ago.

Dr Kearns said she knows of at least 300 industry-funded studies between 1943 and 1972.

‘There is more material than I have the ability to write about,’ she told Daily Mail Online.

‘We will need far more investigators to uncover all of this.’

WHAT WE NOW KNOW ABOUT SUGAR’S LINK TO THE HEART

Today, we are urged to limit our sugar intake as much as possible.

According to FDA regulations, women should have no more than 25g (six teaspoons) of added sugar per day.

That is less than a can of Coca Cola.

Men should have no more than 36g (nine teaspoons) extra.

That equates to a regular Snickers bar.

Sugar, peer-reviewed studies now show, triggers insulin resistance, lower good cholesterol and dangerous bad cholesterol.

It also causes inflammation of the arteries.

These are all direct causes of heart disease.

Dr Glantz concurs.

He was the first to concretely highlight tobacco’s direct links to disease, after receiving more than 4,000 pages of internal Big Tobacco documents in 1994 from an anonymous source. The documents unveiled decades of manipulated research and hidden data proving that cigarettes cause disease.

Dr Glantz went on to publish a book called The Cigarette Papers, which paved the way to more than 1,000 research papers on cigarettes and disease.

Now, he says, we are seeing the same with sugar.

‘The kind of manipulation of research is similar what the tobacco industry does,’ he claims.

‘This kind of behavior calls into question sugar industry funded studies as a reliable source of information for public policy making.’

His previous analysis with Dr Kearns found the SRF had secretly funded a 1967 review playing down evidence linking sucrose consumption to coronary heart disease.

That noted gut microbes may explain why rats fed sugar had higher levels of cholesterol than those fed starch, but dismissed the relevance of animal studies to understanding human disease.

The foundation launched its coronary heart disease research in 1965. The first project was the review published in the New England Journal of Medicine in 1967.

It focused on fat and cholesterol as the dietary cause of coronary heart disease, while downplaying sugar consumption as a risk factor.

Worrying facts: Do you know how much sugar is in your food?

In the new paper the team reports the following year SRF, which changed its name in 1968 to ISRF (International Sugar Research Foundation) launched a rat study called Project 259.

It was to ‘measure the nutritional effects of the (bacterial) organisms in the intestinal tract’ when sucrose was consumed, compared to starch.

The research by W.R.F. Pover of the University of Birmingham, in England, suggested gut bacteria help control sugar’s adverse cardiovascular effects.

Pover also reported findings that might indicate an increased risk of bladder cancer.

Dr Kearns said: ‘This incidental finding of Project 259 demonstrated to ISRF that sucrose vs. starch consumption caused different metabolic effects, and suggested that sucrose, by stimulating urinary beta-glucuronidase, may have a role in the pathogenesis (cause) of bladder cancer.’

The ISRF described the finding in a September 1969 internal document as ‘one of the first demonstrations of a biological difference between sucrose and starch fed rats.’

But soon after ISRF learned about these results – and shortly before the research project was complete – the group terminated funding for the project, and no findings from the work were published.

In the 1960s, scientists disagreed over whether sugar could raise other harmful blood fats called triglycerides more than starch, and Project 259 would have bolstered the case that it could, the researchers argue.

What is more, terminating Project 259 echoed SRF’s earlier efforts to downplay sugar’s role in cardiovascular disease.

Dr Glantz added: ‘Our study contributes to a wider body of literature documenting industry manipulation of science.

‘Based on ISRF’s interpretation of preliminary results, extending Project 259’s funding would have been unfavourable to the sugar industry’s commercial interests.’

Funding was cut off before that could happen.

LAST YEAR’S REPORT: HOW THE SUGAR INDUSTRY FUNDED RESEARCH TO DEMONIZE FAT

The sugar industry paid prestigious Harvard scientists to publish research saying fat – not sugar – was a key cause of heart disease, newly unveiled documents reveal.

At the time, in the 1960s, conflict of interest disclosure was not required.

It meant sugar chiefs could work closely with researchers to re-draft and re-draft their paper until it was ‘satisfactory’ – without having to report their involvement.

The result shaped public health approaches to nutrition for years.

The findings, revealed today in a special report in JAMA Internal Medicine, has sent shockwaves through the research community.

Source: Daily Mail

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Choice of tipple ‘determines different moods’

Herbs and Helpers

Different types of alcohol change and shape your mood in different ways, says a big study into drinking and emotions.

Spirits may make you feel angry, sexy or tearful, while red wine or beer may make you feel relaxed, say researchers.

They questioned nearly 30,000 people aged 18-34 from 21 different countries and present their findings in the journal BMJ Open.

All the respondents drank beer, wine and spirits, and many said each type of alcohol had a different effect on them.

While having a few drinks can be enjoyable, researchers hope their findings will help highlight the dangers of dependent drinking.

Angry outbursts

People build up tolerance to alcohol over time and can end up drinking more to feel the same “positive” effects that they enjoy.

But they also risk getting negative ones too, says researcher Prof Mark Bellis from Public Health Wales NHS Trust.

The anonymous online survey, which recruited respondents via newspaper and magazine adverts and social media, found:

Red wine appeared to make people more lethargic than white wine
Respondents were most likely to report feeling relaxed when drinking red wine or beer
More than 40% said drinking spirits made them feel sexy
Over half said drinking spirits also gave them energy and confidence
But around a third said they felt aggressive when drinking spirits
Drinking spirits was more likely than all other drink types to be associated with feelings of aggression, illness, restlessness and tearfulness.
Men were significantly more likely than women to associate feelings of aggression with all types of alcohol, particularly heavier drinkers.
The findings only show an association, however, and do not explain the reasons for changes.

Prof Bellis said the setting in which the alcohol was consumed was an important factor that the study tried to take into consideration by asking about drinking at home and outside of the home.

“Young people will often drink spirits on a night out, where as wine might be drunk more at home, with a meal.

“There will be an element of expectation too. Someone who wants to relax might choose to have a beer or a glass of wine.”

He said the way different drinks are marketed and promoted might encourage people to select certain drinks to suit different moods, but that this could backfire if it triggers negative emotions.

“People may rely on alcohol to help them feel a certain way. People might drink to feel more confident or relaxed but they also risk other negative emotional responses too.”

Prof Bellis and his colleagues at King’s College London say the findings suggest that dependent drinkers may rely on alcohol to generate the positive emotions they associate with drinking – they were five times more likely to feel energised than low-risk drinkers.

Dr John Larsen from Drinkaware, said: “This study highlights the importance of understanding why people chose to drink certain alcoholic drinks and what effect they expect these drinks will have on them.

“The UK Chief Medical Officers’ guideline for both men and women states that in order to keep health risks from alcohol to a low level it is safest not to be drinking more than 14 units a week on a regular basis.”

That equates to 12 single measures of spirits, six pints of beer or six 175ml glasses of wine a week.

Experts say setting a minimum unit price of 50 pence per unit would help cut alcohol-related deaths.

A minimum price policy will come into force on 1 May 2018 in Scotland.

Legislation to establish a minimum price is currently under active consideration by the National Assembly for Wales and by the Irish Senate. There are no plans yet to do the same in England, although the Home Office says the policy is under review.

Source: BBC

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The Traditional Japanese Herbal Medicine Hachimijiogan Elicits Neurite Outgrowth Effects in PC12 Cells and Improves Cognitive in AD Model Rats via Phosphorylation of CREB

Herbs and Helpers

ORIGINAL: https://doi.org/10.3389/fphar.2017.00850

• 1Department of Neuropharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan

• 2Institute for Aging and Brain Sciences, Fukuoka University, Fukuoka, Japan

• 3Community Medicine Education Unit, Department of Clinical Medicine, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan

Hachimijiogan (HJG) is a traditional herbal medicine that improves anxiety disorders in patients with dementia. In this study, we tested the hypothesis that HJG exerts neurotrophic factor-like effects to ameliorate memory impairment in Alzheimer disease (AD) model rats. First, we describe that HJG acts to induce neurite outgrowth in PC12 cells (a rat pheochromocytoma cell line) like nerve growth factor (NGF) in a concentration-dependent manner (3 μg/ml HJG, p < 0.05; 10-500 μg/ml HJG, p < 0.001). While six herbal constituents of HJG, Rehmannia root, Dioscorea rhizome, Rhizoma Alismatis, Poria sclerotium, Moutan bark, and Cinnamon bark, could induce neurite outgrowth effects, the effect was strongest with HJG (500 μg/ml). Second, we demonstrated that HJG-induced neurite outgrowth was blocked by an inhibitor of cAMP response element binding protein (CREB), KG-501 (10 μM, p < 0.001). Moreover, HJG was observed to induce CREB phosphorylation 20-90 min after treatment (20 min, 2.50 ± 0.58-fold) and CRE-mediated transcription in cultured PC12 cells (500 μg/ml, p < 0.01; 1000 μg/ml, p < 0.001). These results suggest a CREB-dependent mechanism underlies the neurotrophic effects of HJG. Finally, we examined improvements of memory impairment following HJG treatment using a Morris water maze in AD model animals (CI + Aβ rats). Repeated oral administration of HJG improved memory impairment (300 mg/kg, p < 0.05; 1000 mg/kg, p < 0.001) and induced CREB phosphorylation within the hippocampus (1000 mg/kg, p < 0.01). Together, our results suggest that HJG possesses neurotrophic effects similar to those of NGF, and can ameliorate cognitive dysfunction in a rat dementia model via CREB activation. Thus, HJG could potentially be a substitute for neurotrophic factors as a treatment for dementia.

Introduction

Recently, there have been global increases in the number of patients with dementia disorders, such as AD. However, as the pathogenesis of dementia has not yet been fully elucidated, radical curative therapies have not been established. Novel dementia drugs that do not adversely affect ADL are required. In the study of neurodegenerative diseases characterized by neuronal cell death (such as AD), the role of neurotrophic factors has recently drawn attention. Neurotrophic factors are substances that promote neuronal survival, differentiation, and regeneration. Although various trophic factors have been identified, research has indicated particularly important roles for NGF and BDNF (Obara and Nakahata, 2002).

Nerve growth factor is abundant throughout the hippocampus and cerebral cortex, where it stimulates the production, transportation, and secretion of acetylcholine (Ayer-LeLievre et al., 1988; Jonhagen, 2000). Cholinergic neurons, which are NGF susceptible and play an important role in learning and memory, have been shown to be markedly degenerated in the brain of patients with AD. NGF can modulate the metabolism of amyloid precursor protein from amyloidogenic toward non-amyloidogenic processing via binding to the TrkA (Canu et al., 2017). These features guide the design of therapeutic for AD intending to preserve cholinergic function and anti-amyloidogenic activity. However, neurotrophic factors are macromolecular proteins, with an in vivo half-life that is too short to enable delivery to a target tissue or cell. This makes it difficult to directly apply neurotrophic factors as therapeutic agents for AD (Pollack and Harper, 2002; Skaper, 2008). Therefore, attention has turned to developing compounds that show neurotrophic factor-like effects by activating intracellular neurotrophic factor signal transduction pathways and/or promoting biosynthesis of neurotrophic factors. For example, we demonstrated that the Japanese herbal medicine Yokukansan can improve symptoms of dementia (Nogami et al., 2013; Uchida et al., 2013), and its action involves neurotrophic factors such as NGF (Kubota et al., 2013).

In recent years, an association has been reported between neuronal death in AD and decreased activity of CREB (Yamamoto-Sasaki et al., 1999). CREB is a downstream transcriptional regulator of intracellular signaling by neurotrophic factors. CREB is activated through the following three pathways: (1) ERK 1/2; (2) PI3K/Akt; and (3) phospholipase C-γ, all of which lead to CREB phosphorylation (Kaplan and Miller, 2000). In addition to these pathways, AC and the cAMP signaling pathway are also involved in activating CREB and CREB-regulated gene transcription. Activated CREB regulates the transcription of various genes, subsequently inducing spine morphological changes such as neurite outgrowth and branching, and producing long-term potentiation. Additionally, the involvement of CREB in the formation of short-term memories via BDNF induction has been reported (Lonze and Ginty, 2002; Frankland and Bontempi, 2005). Thus, CREB and neurotrophic factor signaling pathways are deeply involved in the formation of both long-term and short-term memory, and are potential targets for drugs to improve cognitive function.

Hachimijiogan (Ba-Wei-Di-Huang-Wan) is an herbal medicine used in China and Japan comprising eight herbs: RR, Cornus fruit, DR, AR, PS, MB, CB, and Aconite root. HJG has traditionally been used to treat diabetes mellitus, hypertension, and nephrotic syndrome. It has also been used to treat dysuria, lumbago, lack of energy, and poor eyesight in older individuals. Furthermore, it has been reported that HJG treatment improved cognitive dysfunction in patients with dementia (Iwasaki et al., 2004). Additionally, several studies, including one of our own, have reported that HJG improves cognitive dysfunction in model animals (Hirokawa et al., 1994, 1996; Moriyama et al., 2017). However, further research is needed to confirm the effects of HJG on cognitive dysfunction. Therefore, to investigate the appropriate clinical application of HJG, we investigated the effects of HJG and its constituent herbs on CREB and neurotrophic factor signaling pathways.

Materials and Methods

Materials

Dry powdered extracts of HJG and its constituents (excluding Cornus fruit and Aconite root) supplied by Tsumura & Co. (Tokyo, Japan) were dissolved in distilled water. HJG is a dried extract of the following eight herbs: RR (6.0 g, the root of Rehmannia glutinosa Libosch var. purpurea Makino, Scrophulariaceae), Cornus fruit (3.0 g, the fruit of Cornus officinalis Siebold et Zucc, Cornaceae), DR (3.0 g, the rhizome of Dioscorea batatas Decne, Dioscoreaceae), AR (3.0 g, the rhizome of Alisma orientale Juzepczuk, Alismataceae), PS (3.0 g, the sclerotium of Poria cocos Wolf, Polyporaceae), MB (2.5 g, the root bark of Paeonia suffruticosa Andrews, Paeoniaceae), CB (1.0 g, the bark of Cinnamomum cassia Blume, Lauraceae), and Aconite root (0.5 g, the root of Aconitum carmichaelii Debeaux, Ranunculaceae).

Nerve growth factor 2.5S was obtained from Life Technologies (Carlsbad, CA, United States). Dimethyl sulfoxide and KG-501 (a CREB inhibitor) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Anti-CREB, anti-phospho-CREB, and anti-rabbit horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, United States). Anti-rabbit β-actin antibodies were obtained from Abcam (Cambridge, United Kingdom).

Donepezil hydrochloride was purchased from Tokyo Chemical Industry (Tokyo, Japan). For animal treatments, HJG and DPZ were dissolved in distilled water. Three weeks after CI, HJG, DPZ, and vehicle (distilled water) were orally administered daily for 7 days.

Cell Culture

PC12 (rat pheochromocytoma) cells were cultured in RPMI-1640 with 5% (v/v) fetal bovine serum, 10% (v/v) horse serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were grown to confluence at 37°C in 5% CO2. The medium was changed two or three times per week. Cells treated with NGF (50 ng/ml) were used as positive controls.

Neurite Outgrowth Assay

PC12 cells (1 × 105 cells/ml) were seeded on collagen-coated six-well plates and cultured for 24 h. Cells were treated with different dilutions of HJG (1-500 μg/ml), or each herb at the concentration based on the original mixing ratio of constituents, and cultured for 72 h. PC12 cells were photographed using an inverted microscope (Olympus, Tokyo, Japan) and phase-contrast objectives. Images of two fields per well were taken with 40-50 cells per field. The length and number of neurites were measured for 20 independent cells per field using Image J 1.44 software (NIH Image Software), and the neurite lengths for each cell were summed. Each experiment was conducted in triplicate.

Western Blotting

Proteins were extracted from cells at appropriate time points after drug treatment using RIPA buffer [50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS] containing a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). Protein concentrations were measured using a BCA Protein Assay Reagent Kit (Thermo Fisher Scientific, Waltham, MA, United States). Proteins were separated on SDS polyacrylamide gels, transferred to polyvinylidene fluoride membranes, and probed with specific antibodies. Immunoreactive polypeptides were detected with chemiluminescence using ImmunoStar LD (Wako, Osaka, Japan).

Luciferase Reporter Assay

PC12 cells (1 × 105 cells/well) were seeded in collagen-coated 12-well plates for 24 h. Cells were co-transfected with the pGL4.29 luciferase reporter vector containing a CRE and pGL4.74 Renilla luciferase vector (Promega) using Lipofectamine 3000 (Life Technologies). Forty-eight hours after transfection, cells were treated with vehicle (0.1% DMSO), HJG, or its constituents for 8 h and harvested using Passive Lysis Buffer (Promega).

Luciferase activities were determined with a Dual-Luciferase Reporter Assay System Kit (Promega) according to the manufacturer’s instructions. We normalized the intensity of luciferase reactions measured in the lysates of transfectants to their Renilla luciferase activity, which was used as an internal control.

Animals

Male Wistar rats weighing 250-300 g were obtained from Kyudo Co., Ltd. (Saga, Japan). Rats were housed in groups of 4-5 per cage at 23 ± 2°C with a relative humidity of 60 ± 10%, and maintained under a 12-h light-dark cycle. Food and water were available ad libitum except during the restricted feeding period. We restricted food intake for rats (8-10 g each day), and their body weight was maintained at ∼80% of free-feeding levels during the eight-arm radial maze tasks. We conducted all procedures relating to animal care and use according to regulations dictated by the Experimental Animal Care and Use Committee of Fukuoka University (No. 1405744).

Preparation of Aggregated Aβ and CI + Aβ Rats

Aggregated Aβ was prepared as previously described (Moriyama et al., 2017). Aβ42 peptides were purchased from AnaSpec Inc. (Fremont, CA, United States), dissolved in HEPES-buffered solution to a final concentration of 10 μM, and incubated at 37°C for 7 days.

Cerebral ischemia + Aβ rats were prepared by combining an intracerebroventricular Aβ injection with transient CI as previously described (Moriyama et al., 2017). Rats were anesthetized with sodium pentobarbital. After exposure and threading of their bilateral common carotid arteries, rats were placed in a stereotaxic frame. The bilateral vertebral arteries were electrocauterized with a bipolar coagulator (MICRO-3D; Mizuho Industrial, Tokyo, Japan). For intracerebroventricular infusion, a guide cannula (0.71 ± 0.02 mm o.d., 0.41 ± 0.02 mm i.d., 13-mm length) was implanted bilaterally into the lateral cerebral ventricles at AP = -0.8 mm, L = ±1.3 mm, and H = 3.3 mm from the bregma. Three days after cannulation, the common carotid arteries were compressed by clips and cerebral circulation was interrupted for 10 min to create CI…

Source: Frontiers

The post The Traditional Japanese Herbal Medicine Hachimijiogan Elicits Neurite Outgrowth Effects in PC12 Cells and Improves Cognitive in AD Model Rats via Phosphorylation of CREB appeared first on Herbs and Helpers – Herbal Services and Solutions | Herbalist | Supplier | Herbs.

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Trendy clogs are terrible for your feet, experts declare: Why HIGH HEELS are better for you than the supposedly ‘comfy’ shoes

Herbs and Helpers

Researchers found that wooden clogs damaged the feet of Dutch farmers who wore them constantly while doing hard manual labor

The shoes can chip the bones of the feet, and wearing them causes repeated ‘micro-traumas’ the researchers say

Modern clogs are mostly made out of more flexible material, but they still cause lasting harm that can even lead to knee, hip and back problems, one New York podiatrist says

If you think that clogs of any kind are a good choice for your feet, think again, experts say.

New research has shown how the traditional wooden variety of the shoe chips away at bones, and a leading podiatrist says that the modern versions are not much better.

Researchers from Western University in Ontario, Canada took a look between the toes of some 500 skeletons from near Amsterdam, and found rare craters had been carved out. Since the bone lesion is unusual, but was so common in these skeletons, the researchers conclude the clogs were to blame.

Modern clogs are made of much more malleable, gentle materials, but New York City podiatrist Dr Suzanne Levine still says they don’t deserve their place on the list of the American Podiatric Medical Association’s accepted footwear options.

Two hundred years ago, wooden clogs were essentially the only footwear fashion in the Netherlands, especially among farming communities.

The carved clunkers were great for keeping farmers’ feet dry and protecting them from incoming cow hooves, but they weren’t exactly doing feet the kinds of ergonomic favors modern shoes are capable of.

Dr Andrea Waters-Rist found that a surprising proportion of her more than 500 skeletons from a farming community in the Netherlands had the same feet problems. The 200-year-old foot bones had similar patterns of osteochondritis dissecans, a relatively condition in which the bone has been chipped away.

Together with her team, Dr Waters-Rist worked out that all of these farmers would have been wearing clogs, day-in and day-out while doing rigorous manual labor. Clogs like theirs are ‘not good at absorbing shock, and constrain the natural movement of your foot,’ she says.

‘If you think about how you walk, you bend your foot and press off the ball of your foot, that’s how you propel yourself forward,’ Dr Waters-Rist says, ‘but a clog-bound foot is not able to do so very naturally. You have to kind of kick the foot forward, and it changes the way we walk.’

I would rather wear heels than clogs Dr Suzanne Levine, podiatrist

The injuries that the farmers sustained were similar to what we see in athletes, such as gymnasts, who repeat that same movements that strain your feet over and over again.

She says that the clogs alone probably wouldn’t have done this damage, but coupled with their daily activities, the shoes inflicted damage that lasted beyond death.

‘The farmers might have been totally asymptomatic…but based on their lifestyle, they probably couldn’t take time off to rest this part of the body,’ says Dr Waters-Rist. ‘They had no choice in the matter: work had to be done, so they went to do it.’

Modern clog-wearers have the luxury of rest, of course, and so long as they can take a break from their questionable fashion decisions, their feet can probably recover.

‘There are a lot of great things about clogs,’ Dr Waters-Rist says, ‘I don’t want them to get a bad rap. They were popular because they were affordable for all, very durable and kept kept feet really dry,’ which was important to farmers working in they boggy Netherlands.

The rubbery descendants of the Dutch clog has become a popular and even recommended choice for people in the healthcare and service industries who have to stand for long stretches of time.

But they shouldn’t be, says Dr Levine.

In fact, ‘I would rather wear heels than clogs,’ she says.

‘So many nurses wear Dansko clogs. They say they accommodate their bunions and corns, but they were originally wood, and made for farmers. The fact that we’re wearing them again is amazing,’ Dr Levine says.

She says wearing clogs for long periods of time can cause fissures in the heels, and stress that travels to the ankles, knees, hips and even back.

‘They’re rigid of sole and have no shock-absorption. You can hear people clogging along, the gait is ridiculous and more importantly the stress fractures and that shearing stretches up the spine’ can cause long term damage, she says.

Dr Levine says that clogs do nothing to support arches, combat gravity or support arches. They feel comfortable because they allow the foot to spread out, but this can lead to plantar fasciitis.

Instead, Dr Levine suggests lace up sneakers for a long day on your feet. She adds that any open-backed shoes should also be avoided, including flip-flops and mules.

‘Having a little heel is so much better for your foot than a very flat shoe,’ Dr Levine says. Flat shoes can lead to flat feet, burning and inflammation under the balls of the feet.

Moreover, there just is not one perfect shoe for every foot. She says that well-constructed shoes made of natural materials are worth the splurge.

In particular, women with wider feet can look to Prada or Steve Madden. If your feet are on the narrow side, try a pair of Louboutins or Zanottis.

But whatever you do, don’t slip into those Danskos.

Source: Daily Mail

The post Trendy clogs are terrible for your feet, experts declare: Why HIGH HEELS are better for you than the supposedly ‘comfy’ shoes appeared first on Herbs and Helpers – Herbal Services and Solutions | Herbalist | Supplier | Herbs.

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