CN114931591A - Application of cotton rose hibiscus leaf extract in medicine for treating neurodegenerative diseases - Google Patents

Abstract

The invention relates to the field of medicinal chemistry, in particular to application of a cotton rose hibiscus leaf extract in a medicament for treating neurodegenerative diseases. The cotton rose hibiscus leaf extract has obvious treatment effect on various caenorhabditis elegans, cells and neurodegenerative diseases of animal models through autophagy induction.

Description

Application of cotton rose hibiscus leaf extract in medicine for treating neurodegenerative diseases

Technical Field

The invention relates to the field of medicinal chemistry, in particular to application of a cotton rose hibiscus leaf extract in a medicament for treating neurodegenerative diseases.

Background

Neurodegenerative Diseases (NDs) are a clinical aging-related disease, and with the accelerated aging of the global population, the morbidity and mortality of neurodegenerative diseases are gradually increased worldwide, which brings huge economic and health burden to the society and individual families. Unfortunately, despite decades of intensive research, there is currently no effective strategy for treating or preventing NDs, and existing treatments only alleviate the symptoms of these diseases or delay the progression of the diseases. Therefore, the search for new effective methods for preventing or treating NDs is urgent.

At present, much research has been carried out on the pathophysiology of NDs, but the understanding of their pathogenesis is still not comprehensive or clear enough. Nevertheless, at the genetic, molecular or cellular level, certain substances or events have been found to recur in patients with NDs, such as protein misfolding and aggregation, neuroinflammation, increased neuronal vulnerability, mitochondrial dysfunction, oxidative stress and iron accumulation. Since familial NDs are often associated with genetic mutations, these mutated genes significantly enhance the aggregation propensity of disease-associated proteins, such as Amyloid Precursor Protein (APP) and Tau protein in Alzheimer's Disease (AD), alpha-synuclein (alpha-synuclein) in PD and hungtintin protein in Huntington's Disease (HD).

More and more studies have shown that protein misfolding and aggregation may play a crucial role in the pathogenesis of NDs. Although there is no apparent similarity in sequence, size, structure, expression level or function between different such classes of NDs-associated aggregated proteins, the processes of misfolding and aggregation of these proteins are very similar.

It has been found that in the brain of NDs, all disease-related proteins misfold from their original state to form intermolecular β -sheet structures ranging from small oligomers to large fibrillar aggregates. Furthermore, a recent breakthrough research finding suggests that misfolded proteins may transfer abnormal pathological proteins between cells and tissues in a prion-like manner via a self-transfer pathway. This finding is important to understand the mechanism of initiation and progression of NDs and suggests that misfolding and transport aggregation of certain proteins are the underlying cause of a particular disease. Thus, targeted clearance of misfolded proteins and reduction of their aggregation appears to be a promising strategy for the treatment of various NDs.

It is well known that the autophagy-lysosomal pathway (ALP) and the ubiquitin-protein system (UPS) are two major proteolytic systems in cells, which play a key role in clearing misfolded proteins, aggregates and damaged organelles. Some recent research results show that the ubiquitin protease system has limited degradation ability to aggregate misfolded proteins, and more importantly depends on autophagy-lysosome pathway to degrade the corresponding protein. Therefore, more and more research is tending to find strategies to treat NDs that can target activation of autophagy.

At present, several compounds capable of enhancing autophagy activity have been discovered and identified, such as curcumin, corynoxeine B, chlorogenic acid, quercetin, resveratrol, etc., which have been shown to exhibit significant neuroprotective effects in cell systems or animal disease models. However, autophagy inducers are still in the initial phase for the treatment of NDs, and most clinical trials of inducers end up with a failure. Although they show promising results in vitro (cellular systems) and in vivo (animal disease models), the majority of autophagy inducing agents are excluded from clinical trials due to toxicity and excessive side effects, limited or ineffective action. Therefore, the development of new safe and effective drugs for treating NDs remains a great challenge, and the strategy for screening novel autophagy inducers must be optimized to improve the possibility of clinical trials and applications.

In recent years, natural products have received increasing attention in the prevention or treatment of NDs. Traditional Chinese Medicine (TCM) provides new possibility for developing potential effective anti-NDs drug pairs due to the advantages of long-term clinical verification, multiple components, multiple targets, high safety, low toxicity and side effects and the like.

Disclosure of Invention

The invention aims to: aiming at the problems of excessive toxicity and side effect, poor treatment effect and even ineffectiveness of an autophagy inducer in the prior art, the application of the cotton rose hibiscus leaf extract in the medicines for treating neurodegenerative diseases is provided.

In order to achieve the purpose, the invention adopts the technical scheme that:

the application of the cotton rose hibiscus leaf extract in preparing the medicines for treating the neurodegenerative diseases is disclosed, wherein the cotton rose hibiscus leaf extract is a cotton rose hibiscus leaf ethanol extract.

The inventor verifies through experiments that the cotton rose hibiscus leaf extract can promote the formation of GFP-LC3 points in GFP-RFP-LC 3U 87 cells, increase the expression of LC3B in PC-12 cells and 3 × Tg-AD mice and reduce the expression of autophagy substrate P62 protein, so that the autophagy induction effect of the cotton rose hibiscus leaf extract in vitro and in vivo is demonstrated, 4 key autophagy related genes are knocked out in an AD nematode model by adopting RNAi bacteria, and the result shows that the neuroprotective effect of the cotton rose hibiscus leaf extract is obviously counteracted. In addition, when autophagy inhibitors 3-MA and Baf blocked autophagy activity, the degradation of APP, Tau and TauP301L proteins by Hibiscus mutabilis leaf extract was reversed in PC-12 cells. Hibiscus mutabilis leaf extract reduced cognitive and memory impairment and inhibited inflammatory responses and apoptosis associated with autophagy activation in 3 × Tg-AD mice. Therefore, the cotton rose hibiscus leaf extract has obvious treatment effect on NDs of various caenorhabditis elegans, cells and animal models through autophagy induction.

As a preferable scheme of the invention, the cotton rose leaf extract is a 65-80 v% ethanol extract of cotton rose leaves. Preferably, the cotton rose leaf extract is a 75% ethanol extract of cotton rose leaves.

As a preferable scheme of the invention, the dosage of the effective components of the medicine prepared from the cotton rose hibiscus leaf extract is more than or equal to 4.0 mg/kg. According to the animal experiment results of mice, when the dosage of the cotton rose hibiscus leaf extract is more than or equal to 50mg/kg, the cotton rose hibiscus leaf extract improves the cognitive and memory disorders of 3 × Tg-AD mice. The dosage of folium Hibisci Mutabilis extract is greater than or equal to 4.0mg/kg by conversion of adult body weight, mouse test body weight and specific surface area.

In a more preferred embodiment of the present invention, the amount of the leaf extract of Hibiscus Mutabilis is 4.0-30mg/kg, and may be 4.5mg/kg, 5mg/kg, 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 15mg/kg, 17mg/kg, 20mg/kg, 25mg/kg, etc.

Furthermore, the dose of the cotton rose leaf extract is 4.0-16.3mg/kg, and experiments show that when the concentration of the cotton rose leaf extract is more than or equal to 50-200mg/kg, the cotton rose leaf extract can effectively improve the cognitive and memory impairment of 3 × Tg-AD mice. Furthermore, the dose of the cotton rose hibiscus leaf extract is 8-16.3 mg/kg; more particularly, the dosage of the cotton rose hibiscus leaf extract is 16.3 mg/kg.

As a preferred scheme of the invention, the preparation method of the ethanol extract of the leaves of the cotton rose comprises the following steps: soaking folium Hibisci Mutabilis in ethanol, and performing ultrasonic treatment; and removing ethanol in the soaking solution after ultrasonic extraction, and then performing freeze-drying to obtain dry powder, namely the ethanol extract of the cotton rose hibiscus leaves.

As a preferred scheme of the invention, 2-5 times volume of ethanol is adopted to soak the leaves of the cotton rose hibiscus for 2-10 days. Preferably, the leaves of Hibiscus mutabilis undergo a crushing treatment.

Preferably, the solution is sonicated during soaking. More preferably, the sonication is performed 1-4 times, e.g. 2, 3 times during.

Preferably, the soaking solution is subjected to solvent removal by evaporation, such as rotary evaporation to dryness, to obtain ethanol extract of Hibiscus Mutabilis.

As a preferred embodiment of the present invention, the medicament is a medicament for preventing and/or treating neurodegenerative diseases. Preferably, the neurodegenerative disease is alzheimer's disease, parkinson's disease or huntington's disease.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

(1) after the treatment of the cotton rose hibiscus leaf extract, the protein level of SQST-1/p62 (shown as GFP) in BC12921 nematodes is reduced, and the accumulation of GFP (shown as LGG-1/LC 3) positive points in DA2123 nematodes is increased, which shows that the cotton rose hibiscus leaf extract has strong autophagy induction activity on transgenic caenorhabditis elegans models.

(2) The cotton rose hibiscus leaf extract is used for obviously promoting the degradation of Abeta, delaying paralysis and improving the food perception disorder of neurons or genetically modified nematodes expressing human Abeta or Tau protein through muscles, which shows that the cotton rose hibiscus leaf extract can effectively improve the symptoms of AD and has the neuroprotective property aiming at the protein aggregation of caenorhabditis elegans.

(3) The treatment of the cotton rose hibiscus leaf extract can obviously prolong the life of wild type N2 nematodes, improve the physique and enhance the resistance to heat, pathogenic bacteria and oxidative stress, which shows that the cotton rose hibiscus leaf extract is one of promising components in the medicines for treating aging and aging-related diseases such as AD, PD and HD.

(4) By using PC-12 cells, the treatment of the cotton rose hibiscus leaf extract is found to obviously inhibit the cytotoxicity of Abeta fibers and APP, Tau and Tau P301L proteins in vitro, and the cognitive and memory functions and AD pathology of 3 × Tg-AD mice are also improved by the administration of the cotton rose hibiscus leaf extract. Therefore, the neuroprotective effect of the cotton rose leaf extract is verified and confirmed in various cells and animal models, and the neuroprotective effect of the cotton rose leaf extract in other models is further verified, so that the feasibility of clinical application is improved.

Drawings

FIG. 1 shows data of the induction of autophagy of caenorhabditis elegans by extracts of leaves of Hibiscus mutabilis;

FIG. 2 is data showing that Hibiscus mutabilis leaf extract exerts neuroprotective effects in models of nematodes of AD, PD and HD;

FIG. 3 is data of Abelmoschus manihot leaf extract to reduce Abeta-induced toxicity in an AD model of transgenic C.elegans;

FIG. 4 shows data of the longevity-extending and pressure-resisting capability-enhancing ability of the Hibiscus mutabilis leaf extract;

FIG. 5 is data showing that Hibiscus mutabilis leaf extract exerts neuroprotective effects in models of nematode worms AD, PD and HD by autophagy induction;

FIG. 6 is data showing that Hibiscus mutabilis leaf extract exerts neuroprotective effects in models of nematode worms AD, PD and HD by autophagy induction;

FIG. 7 shows data of autophagy induction in cells of Hibiscus mutabilis leaf extract;

FIG. 8 is a graph of data showing that Hibiscus mutabilis leaf extract inhibits A β fiber-induced cytotoxicity in cells and promotes degradation of APP, Tau and Tau-P301L proteins;

FIG. 9 shows that Hibiscus mutabilis leaf extract improves cognitive and memory impairment data in mice;

FIG. 10 is data of Abelmoschus manihot leaf extract to reduce A β and Tau symptoms in mice;

FIG. 11 shows data of the inhibition of the activation of NLRP3 inflammasome and the induction of autophagy activation in mice by Hibiscus mutabilis leaf extract.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.

Example 1

1. Preparation of materials

Soaking folium Hibisci Mutabilis in 75% ethanol twice the volume of folium Hibisci Mutabilis for 7 days, wherein the folium Hibisci Mutabilis is dried and pulverized, and performing ultrasonic treatment for 2 times, each for 30 min. After ultrasonic extraction, the soak solution is transferred to an evaporation dish or a rotary evaporator to remove most of the ethanol solvent. Then, the extract is made into dry powder by a freeze drying mode, namely the ethanol extract of the leaves of the cotton rose. Storing at-80 deg.C for use.

2. Testing

2.1 caenorhabditis elegans: strain, culture and synchronization

Caenorhabditis elegans is a relatively simple invertebrate with small size, short life span, and rapid cycle

3 days), high reproduction rate, easy culture, low cost, definite cell line and dissectionStructure and the like. More importantly, it has a complete and easily controlled nervous system, consisting of 302 neurons and approximately 5000 chemical synapses, giving it the ability to respond neurotoxically. Although transgenic mice, as more complex mammals, better mimic and represent the actual process of human NDs, they are slow in cycle, expensive and unsuitable for drug screening. Thus, nematodes have become a simple and reliable animal model in research in the fields related to neurodegenerative diseases and aging.

Wild type nematode strain N2, transgenic nematode strain BC12921[ rCes T12G3.1:: GFP + pCeh361], DA2123 [ lgg-1p:: GFP:: lgg-1+ rol-6(su1006) ], CL2331[ myo-3p:: GFP:: A-Beta (3-42) + rol-6(su1006) ], CL4176[ myo-3p:: A-Beta (1-42): let-8513 'UTR) + rol-6(su1006) ], CL2335[ pCL45 (Psnb-1:: Abeta 1-42::3' o::::::: + ld: + 2:: UTR GFP ], CL2122[ (ppd30.38) unc-54 (vector) + (L26) mtl-2: BR 70:: 70: 34-75: 34. sup.: stem-7: 34. sup.: 32. sup.: 34. sup. wt.% GFP, M35. sup. 3538) unc-54 (vector) + (L26), McMR 5232. mCP 5[ mCP ] mCP 5 ]: 5232:: 32: (mCP ] mCP 5:: 32: (mCP) and 75: (mCP) mCP 7: [ 10: (34: (mCP) mCP 7: (34:: 34. sup.: 9) (III) mCP 7: 34. sup. 34. sup. 34. sup. 34. sup. 34. sup. 34. sup. 34. sup. 34. sup. wt.: 9) stem K), NL5901[ unc-54p:: alphasynuclein:: YFP + unc-119(+) ], BZ555[ dat-1p:: GFP ], AM141[ unc-54p:: Q40:: YFP ], CF1553[ (pAD76) sod-3p:: GFP + rol-6(su1006) ], SJ 0[ hsp-6:: GFP ] and CL2166[ (pAF15) gst-4p:: GFP:: NLS ] all nematode strains were purchased from the C.elegans Genetic Center (CGC). Unless otherwise noted, caenorhabditis elegans were grown on standard Nematode Growth Medium (NGM) using E.coli OP50 as a food source in a constant temperature and humidity chamber at 20 ℃ and 60% humidity.

2.1 synchronization and culture of nematodes

All prior to the trial larvae at stage L1 were obtained by synchronization, as follows: eggs were separated from egg-laying worms using bleaching solution (0.5M NaOH + 1% NaClO), washed at least 3 times with M9 buffer, and then incubated overnight in M9 buffer. Subsequently, the synchronized L1 stage larvae were transferred to NGM plates containing OP50 for culture, and when the nematodes grew to L4 stage, they were transferred to fresh NGM plates supplemented with 5 mg/L5-fluoro-2' -deoxyuridine (FUDR) for continued culture to inhibit post-oviposition hatching of adult nematodes.

2.2 quantitative analysis of SQST-1/p62: GFP and GFP: LGG-1 fluorescence in the nematodes DA2123 and BC12921

Transgenic nematode strains DA2123 and BC12921 were used in this study to monitor nematode autophagy events as follows: the synchronized L1 stage nematode DA2123 and BC12921 larvae are spread on a NGM flat plate containing cotton rose leaf extract or Rap, cultured in an incubator at the temperature of 20 ℃ for 48h, then the nematodes are transferred to a microscope slide containing 100mM sodium azide for anesthesia, the microscope slide is lightly covered with a cover glass, and at least 30 GFP fluorescence pictures are observed and taken under an upright fluorescence microscope. ImageJ software was used to count the number of positive fluorescent spots in suture cells or intestinal cells of the DA2123 nematodes in the fixed area and to quantify the fluorescence intensity of the BC12921 nematodes. In addition, the expression of GFP protein was verified by detecting PE-GFP:LGG-1, GFP:LGG-1 and SQST-1/p62 by Western blotting.

2.3 assay of A.beta. (3-42) aggregation in CL2331 nematodes

The CL2331 strain was used to analyze the aggregation of nematode A.beta.3-42. Briefly, synchronized L1 larvae were treated with cotton rose hibiscus leaf extract at 15 ℃ to reach L4 late stage larvae. Then transferred to NGM plates containing Fudr and Hibiscus Mutabilis leaf extract to prevent offspring hatching, and cultured at 25 deg.C for 4 days. After treatment, nematodes were collected in M9 buffer, transferred to microscope slides containing 100mM sodium azide for anesthesia, covered gently with a cover slip, observed under an upright fluorescence microscope and photographed for at least 30 GFP fluorescence. ImageJ software was used to measure the number of A β (3-42) LGG-1 positive fluorescent spots in the fixed area.

2.4 test for A β -induced paralysis in CL4176 and CL2006

Transgenic nematode strains CL4176 and CL2006 which specifically express the human Abeta 1-42 protein through muscles are used for respectively carrying out acute or chronic paralysis experiments. The specific operation is as follows:

CL 4176: after the L1-stage nematodes are maintained and cultured in an incubator at 15 ℃ for 36h, the nematodes are quickly transferred to an incubator at 25 ℃ for further culture for 30h, and then the nematode state is observed under a microscope at any time. When more nematodes on the plate enter a paralyzed state, photographing and counting are started. The nematode can not move due to external mechanical stimulation (such as the nematode needle lightly touching the body) or head movement, and the body can not move, namely, the nematode is paralysis.

CL 2006: after the L1-stage nematodes were maintained in the 20 ℃ incubator for 52h, they were transferred to plates containing Fudr to prevent offspring hatching, and then they were cultured in the 20 ℃ incubator with fresh NGM plates being changed every two days. When the nematodes begin to enter the paralyzed state, the paralyzed number of the nematodes begins to be recorded, and statistics is carried out once every 24 hours.

2.5 food perception test

Food-sensing behavior analysis was performed to assess nematode neurological function. Nematodes move rapidly in plates without bacteria and slowly in bacterial turfs to feed themselves more efficiently. However, when the nervous function of nematodes is disrupted, their movement in bacterial lawns is slowed. NGM plates with or without OP50 will be prepared in advance, and the nematodes treated with cotton rose hibiscus leaf extract will then be transferred to plates with or without OP 50. To avoid stress, the nematode was allowed to stand for 90 seconds and then counted for the number of body bends in 20 seconds. The food perception capability is reflected by the deceleration rate, and the specific formula is as follows:

deceleration rate (N) _{Food-free food} -N _{With food} )/N _{No food} (ii) a N represents the number of body bends in 20 s.

2.6 body bending test of NL5901 and AM141 nematodes

The synchronized L1 stage transgenic nematode strain NL5901 or AM141 was cultured on NGM plates with or without Hibiscus mutabilis leaf extract. On days 5 and 10 after adulthood, the treated nematodes were transferred to M9 buffer and left for 120 seconds to avoid interference with stress-related behavior. The number of body bends over 20 seconds was then observed and recorded under a stereomicroscope, while a representative video of nematode swimming was taken with Bandicam software.

2.7 fluorescent quantitation of alpha-synuclein and polyQ40 aggregates

NL5901 and AM141 nematode strains are used for analyzing the aggregation of alpha-synuclein and polyQ40 protein in the nematodes. Briefly, synchronized transgenic nematode strains NL5901 or AM141, stage L1, were cultured on NGM plates with or without cotton rose hibiscus leaf extract. After treatment, nematodes were collected in M9 buffer, transferred to microscope slides containing 100mM sodium azide for anesthesia, covered gently with a cover slip, observed under an upright fluorescence microscope and photographed for at least 30 GFP fluorescence. ImageJ software was used to measure the fluorescence intensity of each nematode α -synuclein:: YFP or polyQ40:: LGG-1.

2.8 treatment with 6-OHDA and quantitative determination of DA neurodegeneration

Caenorhabditis elegans BZ555 strain expressing GFP in DA neurons was treated with 6-OHDA to establish a PD model of DA neurodegeneration, in particular as follows: the L3 stage nematodes were transferred to NGM broth containing 50mM 6-OHDA, 10mM ascorbic acid and OP50 for 1h with gentle mixing every 10 minutes. After incubation, the cells were washed 3 more times with M9 buffer until the liquid was substantially clear or nearly clear. The nematodes were then transferred to NGM plates containing extracts of leaves of Hibiscus mutabilis or levodopa, cultured at 20 ℃ for 72h, collected with M9 buffer, transferred to microscope slides containing 100mM sodium azide for anesthesia, coverslipped gently, observed under an upright fluorescence microscope and photographed for at least 30 GFP fluorescence photographs. The GFP fluorescence intensity in the head fixation region of each nematode was measured using ImageJ software.

2.9 Life measurement

Prior to the experiment, nematodes had to be stably cultured for more than 3 generations without starvation. Synchronized L1 larvae were maintained in culture on NGM dishes for 52 hours to grow to late L4 or early adult stages. The nematodes were then transferred to fresh NGM dishes containing 200. mu.g/mL Hibiscus mutabilis leaf extract and 5mg/LFUDR to prevent hatching of the eggs. We define the first day of starting treatment with the extract of leaves of Hibiscus mutabilis as day 0 of the life test of adult nematodes and transfer the nematodes to corresponding fresh plates every 1-2 days during the period from day 0 to day 10 to ensure the efficacy of the drug. The life statistics were performed starting from the moment of normal nematode death (not more than day 10 at the latest) and once a day at a fixed time. If the body or head of the nematode is lightly stimulated with platinum wire, the nematode will not respond to any physical stimulus and will be defined as dead. For nematodes that escape the plate, rupture of the reproductive tract or spill of intestinal contents, statistics were not included. At least 60 nematodes per group need to be kept in a minimum statistical amount per life test.

2.10 quantification of pharyngeal and crawling function analysis

In the pharyngeal twitch and speed test, wild type N2 nematodes were treated with or without extracts of Hibiscus mutabilis according to the longevity test method. Treated nematodes were observed and analyzed under a stereomicroscope on days 5 and 10 of adulthood.

For pharyngeal function analysis, one pharyngeal pumping was defined as one contraction of the posterior sphere/mill, and the number of pharyngeal pumping events for the nematodes was counted in 20 seconds. And observing the crawling state of the nematodes under a stereoscopic microscope, and simultaneously acquiring the motion video of the nematodes by using Bandicam software. Mean and maximum nematode velocities were analyzed and quantified by ImageJ and wrMTrck Batch.

2.11 RNAi interference by caenorhabditis elegans

Single clones of RNAi bacteria were transferred to LB medium/ampicillin (50. mu.g/mL), shaken continuously overnight at 37 ℃ and induced to synthesize dsRNA by addition of isopropyl thiogalactoside (IPTG,5 mM). In order to inhibit 4 key autophagy-related genes (unc-51, bec-1, vps-34 and lgg-1) in nematodes, synchronized L1 nematodes were plated on NGM plates containing extracts of leaves of Hibiscus mutabilis and RNAi bacteria, and then maintained at 20 ℃ for culture, and nematodes were collected at corresponding time points to determine various indicators.

2.12 RNA extraction and qRT-PCR

Total nematode RNA was extracted using the RNAioso Plus reagent (TaKaRa) according to the manufacturer's instructions. Then, the DNA was transcribed with One-Step cDNA Synthesis Kit (Takara, 6110A), and 1. mu.g of total RNA was used as a template for reverse transcription into cDNA. The qRT-PCR was performed on a Bio-Rad CFX96 real-time system and a C1000 thermal cycler (Bio-Rad, CA, USA) using SYBR Green Dye (Takara) as a fluorescent probe. The transcription level of the gene was normalized by the transcription level of cdc 42. The primers used for qRT-PCR are listed in Table S1.

2.13 cell culture

PC-12 cells were obtained from American type culture Collection (ATCC, Rockville, Md., USA) and were selected for evaluation of the neuroprotective effects of extracts of Hibiscus mutabilis leaves. Stable GFP-RFP-LC 3U 87 cells were generously given by Baker of Xiaoming (university of Australian science, Australian, China). In this, PC-12 cells were cultured in DMEM basal medium containing 10% Fetal Bovine Serum (FBS) (Gibco, Rockville, Md., USA), 50U/mL penicillin and 50. mu.g/mL streptomycin (Invitrogen, Scotland, UK). GFP-RFP-LC 3U 87 cells were cultured in α -MEM basal medium supplemented with 10% FBS, 50U/mL penicillin, and 50 μ g/mL streptomycin. All cells were maintained in an incubator with 5% carbon dioxide, 75% humidity, 37 ℃.

2.14 preparation of Abeta (1-42) cellulose

1mg of lyophilized A β (1-42) peptide was dissolved in hexafluoroisopropanol (HFIP, Sigma), then re-lyophilized and stored at-80 ℃. The stored monomeric peptides were each dissolved in dimethyl sulfoxide (DMSO, Sigma) to a final concentration of 2mM and then used after 7 days of incubation at 37 ℃. The stock was finally diluted in sterile phosphate buffered saline (PBS, Sigma) at a concentration of 20. mu.M in the culture medium.

2.15 cell viability assay

Cell counting kit-8 (CCK-8, MedChemexpress, Monmouth Junction, NJ, USA) was used to detect cell viability. By 5X 10 ³ Density per well cells were seeded into 96-well plates, and after cell treatment was complete, the old medium was removed and fresh medium containing 10 μ L CCK-8 solution was added to each well of the plate. Subsequently, the plate was gently mixed for 30 seconds and cultured at 37 ℃ for 2 to 4 hours in a cell culture chamber containing carbon dioxide at a concentration of 5%. Finally, the absorbance (OD) value of the solution was measured at 450nm using a rotation 3 imager (BioTek, VT Lab, USA). Cell viability was calculated using the following formula:

cell survival (%) ═ OD value _{Drug treatment} OD value _Control ×100。

2.16 quantitative analysis of GFP-LC3 fluorescent spots

The stabilized GFP-RFP-LC 3U 87 cells were seeded onto coverslips in 6-well plates. Treating cells with folium Hibisci Mutabilis extract, and polymerizing with 4%Formaldehyde (PFA) cells were fixed at room temperature for 20 minutes and then washed 2 times with PBS. After the slides were air dried, FluorSave was used ^TM Mounting medium (Calbiochem, San Diego, CA, USA) was mounted as a sheet. Then, the CCD digital camera is mounted at Spot RT3 ^TM Representative images of cells expressing the fluorescent spot of LC3 were observed and taken under an ECLIPSE 80i fluorescence microscope (Diagnostic Instruments, inc., Melville, NY, USA) and the number of GFP-LC3 spots per cell was measured in GFP positive cells. At least 150 cell counts were selected from 3 randomly selected regions.

2.17 Thioflavin T fluorescence assay

Freshly prepared stock solutions of Abeta (1-42) (dissolved at 2mM in DMSO) were removed and diluted to a final concentration of 20. mu.M with 100. mu.L of PBS buffer with or without Hibiscus mutabilis leaf extract. The mixture was then transferred to an incubator at 37 ℃ for 24 hours, 48 hours or 72 hours. After treatment, 10. mu.L of the mixture was transferred to a fluorescent 96-well plate, 190. mu.L of ThT solution (20. mu.M) was added thereto and mixed well, and then the mixture was transferred to an incubator at 37 ℃ for further incubation for 1 hour. After incubation was complete, the fluorescence intensity of each set of ThT solutions was measured at an excitation wavelength of 440nm and an emission wavelength of 470nm using a Cytation 3 microplate reader.

2.18 animals

3 × Tg-AD mice carrying the human mutant genes APPswe, Psen1 and TauP301L (strain: B6; 129-Tg [ APPSwe, tauP301L ] Psen1tm1Mpm/Mmjax, No.: 34830-JAX) were purchased from Jackson laboratories (Bar Harbor, ME, USA). 3 × Tg-AD mice (model mice) and C57BL/6J mice (wild-type normal mice) were housed in an SPF-rated animal house (humidity, 60%; temperature, 20-24 ℃) given a 12 hour light/dark cycle and freely available food and water. Male and female mice (20-22 g) of similar body weight were randomly selected and assigned a number and divided for study prior to dosing intervention. The groups in the study comprise a normal control group (normal mice are subjected to intragastric perfusion by normal saline), a model group (3 XTG-AD mice are subjected to intragastric perfusion by normal saline), a low-dose cotton rose hibiscus leaf extract group (3 XTG-AD mice are subjected to intragastric perfusion by cotton rose hibiscus leaf extract with the weight of 50mg/kg per kilogram), a medium-dose cotton rose hibiscus leaf extract group (3 XTG-AD mice are subjected to intragastric perfusion by cotton rose hibiscus leaf extract with the weight of 100 mg/kg per kilogram) and a high-dose cotton rose hibiscus leaf extract group (3 XTG-AD mice are subjected to intragastric perfusion by cotton rose leaf extract with the weight of 200mg/kg per kilogram). The mice of the normal control group, the model group and the treatment group of the cotton rose hibiscus leaf extract are irrigated with the same volume of normal saline or cotton rose hibiscus leaf extract solution once a day at 5-6 pm within 60 consecutive days, and the treatment sequence is random. Prior to sacrifice, the cognitive function of the mice was assessed using the Y-maze (YM) and Morris Water Maze (MWM) tests following the last dose of treatment. Subsequently, all mice were anesthetized with 10% chloral hydrate (4 ml/kg) and decapitated to separate brain tissue. Brain tissue from mice was stored at-80 ℃ for Western blotting experiments or fixed with 4% Paraformaldehyde (PFA) for immunohistochemistry experiments. All animal care and experimental procedures involved in this study were approved by the Animal Ethics Committee (AEC) of the southwest medical university.

2.19Y-maze test

The spontaneous alternation test was carried out in a symmetrical white plexiglas Y-shaped maze having three arms (20 cm long by 10 cm wide by 20 cm high) at an angle of 120 DEG, each of the three arms being designated A, B, C. Mice were placed distal to the a-arm and allowed to explore the maze for 5 minutes. The trajectory of the mouse was recorded with a camera mounted above the maze and a DigBehv animal behavior analysis system (shanghai jiliang software technologies ltd, shanghai, china). The percentage of the number of alternations (the number of entries into one arm different from the first two) was calculated using the following formula. (Alternatives/(Arm Entries-2)). 100.

2.20 Moris Water maze test

The Morris Water Maze (MWM) assay was used to evaluate spatial learning and memory performance in 3 × Tg-AD mice. The MWM system was equipped with a circular dark grey water basin (120 cm diameter; 50 cm height) filled with water and maintained at a temperature of 22-24 ℃. The pool was divided into four quadrants, with an escape platform (8 cm diameter) centered 2 cm below the water surface in the center of the third quadrant. Each mouse required training twice daily for 5 consecutive days prior to testing. During training, each mouse entered the water from one of the four quadrants and was allowed to find the platform and dwell on it for 10 seconds within 60 seconds. After training, the platform was removed, mice were placed in the water from the same quadrant, and the swimming trajectory of the mice within 60 seconds and the number of mice entering the quadrant of the original platform were recorded using the visotack rodent behavior analysis system (shanghai new raney information technology limited, shanghai, china). In addition, the time of the mouse in the quadrant of the original platform, the frequency of passing through the quadrant of the original platform and the distance of the mouse in the quadrant of the original platform are all measured by the system.

2.21 Western blot analysis (Westernblotting)

After the sample is treated, a sample of brain tissue from cells, nematodes or mice is lysed with RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was measured by Quick Start ^TM Bradford 1 × Dye protein assay Reagent (Bio-Rad, Calif., USA). A mixture of 30. mu.g of total protein per sample was aspirated and added to SDS-PAGE for separation. Subsequently, the proteins separated on the gel were transferred to a polyvinylidene fluoride (PVDF) membrane. Thereafter, PDVF membranes were incubated with 5% skim milk at room temperature for 1 hour, followed by primary antibody at 4 ℃ overnight, followed by HRP-conjugated secondary antibody (1:2000) at room temperature for 1 hour, and proteins were detected with specific antibodies as listed in Table S2. Finally, the Western blot bands were visualized using UltraSignalTM ECL Western blot detection reagent (4A Biotech, Inc., Beijing, China) and detected using the ChemiDoc MP imaging System (Bio-Rad, Berkeley, Calif.). Finally, the intensity of the bands representing the relative expression level of the protein was quantified using ImageJ software.

2.22 immunohistochemistry

After the whole mouse brain was fixed in 4% Paraformaldehyde (PFA) for 48 hours, it was dehydrated in 95% ethanol and then frozen in Optimal Cutting Temperature (OCT) complex embedding medium. Subsequently, the frozen tissue was cut into a sheet including the hippocampus and fixed on a glass slide. The sections were deparaffinized and rehydrated by a series of incubations in xylene and ethanol. After reconstitution, antigen retrieval was performed in a humidity chamber using EDTA solution at pH9.0 and citric acid buffer at pH 6.0. The tissue sections were then cooled at Room Temperature (RT), and the antigen-reconstituted sections were subsequently incubated in 0.3% H2O2 in methanol for 30 minutes to quench the activity of endogenous peroxidase. Then, the extracted antigen sections were washed, incubated with the appropriate primary antibody at 27 ℃ for 1 hour, washed of excess antibody, and incubated with the appropriate secondary antibody at 27 ℃ for 30 minutes. After the secondary antibody incubation, sections were visualized with 3-3 Diaminobenzidine (DAB). Representative images were taken with an optical microscope (Nikon, japan) and optical density values representative of protein expression were analyzed with ImageJ software.

2.23 Elisa assay of A.beta.1-42

The expression level of A.beta.1-42 in the brain and serum of 3 × Tg-AD mice was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Rexin organism, RX 203110M). Briefly, mouse brain tissue and serum samples were prepared according to the protocol described in the ELISA kit. The supernatant was then processed in flat bottom 96 well ELISA plates according to the manufacturer's instructions and the absorbance OD was measured at 450nm using the rotation 3 microplate reader. The levels of A β (1-42) in brain and serum of 3 × Tg-AD mice are expressed in pg/mL. All experiments were repeated at least three times.

2.24 statistical analysis

Data expressed as mean ± Standard Deviation (SD) were analyzed using GraphPad Prism 6.0 software (San Diego, CA, USA), using one-way analysis of variance, followed by Tukey post-tests. The results shown are from at least three independent experiments. P <0.05 was considered statistically significant.

3. Results

3.1 Hibiscus mutabilis leaf extract induces autophagy of caenorhabditis elegans

A transgenic nematode strain DA2123 is used as a high-throughput screening model, and natural products with strong autophagy induction activity are screened from a Chinese herbal medicine library. It was reported that when autophagy was activated, LGG-1/ATG8 was sequestered on the membrane of autophagosomes and aggregated into spots, and thus the expression of this protein could serve as a marker event for the occurrence of autophagy. The treatment of the cotton rose hibiscus leaf extract (FHME) significantly increased the number of LGG-1: GFP positive spots in DA2123 nematode seam cells and intestinal cells, which is comparable to Rap, a typical potent autophagy activator, indicating that the cotton rose hibiscus leaf extract can effectively induce autophagy of nematodes (a and B in fig. 1). Meanwhile, Western blotting results showed that the ratio of Phosphatidylethanolamine (PE) -bound GFP:: LGG-1 (PE-GFP:: LGG-1) to non-lipidated GFP:: LGG-1 was significantly decreased (FIG. 1, E).

During autophagy, SQST-1/p62 protein, a substrate specific for autophagy, interacts with LC3 protein to infiltrate autophagosomes and is degraded in lysosomes, thereby indirectly reflecting the activity of autophagy. By utilizing the principle, the autophagy activity of the cotton rose hibiscus leaf extract is verified by utilizing a transgenic nematode strain BC12921 expressing SQST-1/p 62:GFPfusion protein in vivo. Compared with the control group, after 48 hours of treatment of the BC12921 nematodes by the cotton rose hibiscus leaf extract, the SQST-1/p62 shows that the GFP protein is obviously degraded and has statistical significance (C and D in figure 1). In addition, the expression level of GFP protein in BC12921 bodies is detected by Westernblotting, and the result shows that the extract of leaves of cotton rose hibiscus also reduces the SQST-1/p62: (F in figure 1) the protein level of GFP. qRT-PCR analysis showed that the treatment of the leaf extract of Hibiscus mutabilis significantly increased the mRNA levels of autophagy-related genes including unc-51 and vps-34 (G in FIG. 1) in the wild-type N2 nematodes. The results show that the cotton rose hibiscus leaf extract can induce autophagy of caenorhabditis elegans.

3.2 Hibiscus mutabilis leaf extract reduces Abeta and Tau-induced toxicity in AD model of transgenic C.elegans

Enhancing autophagy plays an important role in the prevention of various neurodegenerative diseases, including AD, PD and HD. In a wide range of past reports, a β plaque deposition is thought to play an important role in the pathogenesis of AD. Therefore, firstly, the inhibition effect of the hibiscus mutabilis leaf extract on the aggregation of A beta in the nematode body is researched by utilizing the transgenic nematode strain CL2331 for expressing human A beta (3-42) GFP protein in the somatic muscle cells of the nematode body in a temperature sensitive way. As shown in A and B in figure 3, the treatment of the cotton rose hibiscus leaf extract significantly reduced the number of Abeta (3-42) -GFP positive spots in CL2331 nematodes, indicating that the cotton rose hibiscus leaf extract can degrade Abeta (3-42) aggregates in vivo. In addition, paralysis induced by A.beta.aggregation toxicity was detected using transgenic CL4176 worms temperature-sensitively expressing A.beta. (1-42) protein in somatic muscle cells. As shown in A and C in figure 2, the paralysis rate of CL4176 nematodes is obviously reduced by 100-mL of the Hibiscus mutabilis leaf extract with 200. mu.g/mL, and the best effect is shown by 200. mu.g/mL of the Hibiscus mutabilis leaf extract. Therefore, 200 μ g/mL of Hibiscus mutabilis leaf extract was selected as the optimal therapeutic concentration in subsequent nematode experiments.

In order to prove the protection effect of the cotton rose hibiscus leaf extract on A beta-induced toxicity, transgenic nematode CL2006 which directionally expresses A beta (1-42) protein in body wall muscle cells is adopted. It was found that treatment with extracts of cotton rose hibiscus leaves also significantly delayed paralysis of the CL2006 nematodes (fig. 3D). Furthermore, we tested A β -induced impairment of food perception using transgenic nematode strain CL2335, which expresses A β (1-42) protein temperature-sensitively in pan-neurons, and its control strain CL 2122. As expected, the CL2335 nematodes showed a defect in food perception, mainly in slowing down the rate of decline. However, the treatment with hibiscus mutabilis leaf extract effectively protected this defect (B in fig. 2). In addition, a transgenic nematode strain BR5270 expressing the human Tau protein in pan-neurons and its control strain BR5271 were used to evaluate the neurotoxicity of Tau. Food perception test results showed that the neuron-damaged BR5270 nematodes showed a defect in food perception, which was significantly improved by treatment with the extract of cotton rose leaves, as evidenced by a reduction in speed (C in fig. 2). Taken together, the results indicate that hibiscus mutabilis extract exhibits potent neuroprotective effects on abeta and Tau-induced c.

3.3 Hibiscus mutabilis leaf extract inhibits alpha-synuclein pathology and inhibits DA degeneration in nematode PD model

There is increasing evidence that impairment of autophagy function is closely associated with PD, and enhancement of autophagy may improve the symptoms of PD. In this study, whether extracts of Hibiscus mutabilis improve the pathological state of alpha-synuclein was examined by using transgenic NL5901 nematodes expressing human alpha-synuclein and Yellow Fluorescent Protein (YFP) fusion proteins in somatic muscle cells. The results showed that the YFP intensity was significantly decreased in NL5901 nematodes treated with the cotton rose leaf extract at 5 and 10 days (D and E in fig. 2), indicating that the cotton rose leaf extract can inhibit the accumulation of alpha-synuclein. During senescence, the locomotor capacity of NL5901 nematodes decreases with aggregation of α -synuclein. However, treatment with cotton rose hibiscus leaf extract significantly improved the motor ability defect of NL5901 nematodes, which is mainly reflected in the number of body bends of the nematodes within 20 seconds (F in fig. 2). The results show that the treatment of the cotton rose hibiscus leaf extract can improve the pathology of alpha-synuclein in the caenorhabditis elegans PD model.

In addition, neurotoxin-induced degeneration of DA neurons is another important pathological hallmark of PD. Therefore, the dopamine neuron is labeled by the specific expression of GFP of the transgene nematode BZ555 under the action of the dat-1 promoter, and can be directly observed under a fluorescence microscope. Then 6-OHDA is used for inducing selective apoptosis of DA neurons of BZ555 worms, so that another PD model is established, and whether the cotton rose hibiscus leaf extract can inhibit DA neuron damage or not is further researched. The results indicate that the GFP intensity of the 6-OHDA-treated BZ555 nematodes is significantly lower than that of untreated BZ555 nematodes, indicating that 6-OHDA causes severe damage to DA neurons. However, treatment with cotton rose hibiscus leaf extract significantly inhibited 6-OHDA-induced neurological damage, with effects similar to levodopa (G and H in fig. 2). It has also been reported that 6-OHDA-induced nematodes also cause a dysfunction in food perception due to DA neuron damage. Therefore, it was further investigated whether the extract of leaves of Hibiscus mutabilis could improve the food perception disorder of 6-OHDA-induced BZ555 nematode. As shown by I in FIG. 2, the 6-OHDA-induced deceleration rate of BZ555 nematode was significantly reduced to 13% compared to the control group, while the treatment with the extract of Hibiscus mutabilis leaf or L-Dopa showed significant recovery of the deceleration rate to 50.3% and 47.2%, respectively. The results show that the extract of leaves of cotton rose hibiscus exerts a neuroprotective effect in the PD model of caenorhabditis elegans.

3.4 Hibiscus mutabilis leaf extract inhibits the aggregation of polyQ40 and improves nematode motility

The transgenic caenorhabditis elegans AM141 is utilized to research whether the Hd-related pathological and behavioral defects can be improved by the Hd leaf extract. This nematode strain specifically expresses a polyglutamine repeat (polyQ40) driven by the unc-54 promoter in muscle cells and mimics the HD-like phenotype. As shown by J, K in figure 2 and E in figure 3, the polyQ40 aggregates in the AM141 nematodes gradually accumulated with the increase of age, but the number of polyQ40 aggregates was significantly reduced on the 5 th and 10 th days after the treatment with the cotton rose hibiscus leaf extract, which suggests that the cotton rose leaf extract can inhibit the expression of polyQ40 in the AM141 nematodes. In addition, the movement ability of AM141 worms was significantly improved by treatment with the extract of cotton rose leaves, which significantly improved the movement ability of AM141 nematodes (L in FIG. 2). The results show that treatment with Hibiscus mutabilis leaf extract can alleviate polyQ40 aggregation and improve behavioral deficits in the caenorhabditis elegans HD model.

3.5 Hibiscus mutabilis leaf extract prolongs the life and improves health of wild type nematode N2

Aging is a major risk factor for the development of neurodegenerative diseases. Therefore, the effect of the cotton rose leaf extract on caenorhabditis elegans senescence was examined. Compared with the control group, the extract of leaves of Hibiscus mutabilis can prolong the life of wild type N2 nematode, with average life prolonging rate of 11.1% (FIG. 4A). Previous studies have shown that the age-related phenotypes currently known, such as pharyngeal pumping behavior and motor capacity, decrease with age, and that lipofuscin accumulates with age. Therefore, whether the cotton rose hibiscus leaf extract improves nematode health was investigated by assessing feeding and locomotor ability of wild-type N2 nematodes. As shown in B in figure 4, the treatment of the N2 nematodes by the cotton rose hibiscus leaf extract can significantly improve the pharyngeal pumping rate on the 5 th day and the 10 th day, which indicates that the cotton rose hibiscus leaf extract delays the decline of the pharyngeal muscle function with age. Furthermore, by measuring body curvature, mean velocity and maximum velocity within 20s of nematodes, we found that the cotton rose hibiscus leaf extract also significantly enhanced the locomotor ability of the N2 nematodes, young (5 days old) and old (10 days old) (C-E in fig. 4). In addition, treatment with Hibiscus mutabilis leaf extract significantly reduced the intestinal lipofuscin deposition of N2 worm (F in FIG. 4). In conclusion, the cotton rose hibiscus leaf extract can prolong the life of caenorhabditis elegans and delay senescence-associated phenotypes, which indicates that the cotton rose hibiscus leaf extract has anti-aging effect.

3.6 the extract of leaves of Hibiscus mutabilis enhances the anti-stress ability of caenorhabditis elegans

There is increasing evidence that resistance to external stimuli is closely related to the aging process and neurodegenerative diseases. Caenorhabditis elegans, which have been extended in life by genetic or non-genetic manipulation, generally exhibit greater resistance to stress. Therefore, we investigated whether the extract of leaves of Hibiscus mutabilis would improve the resistance of caenorhabditis elegans to heat, pathogenic bacteria and oxidative stress. As shown in G of FIG. 4, the survival rate of N2 nematodes was significantly improved by the treatment of Hibiscus mutabilis leaf extract under the heating condition of 35 deg.C. Molecular chaperones of Heat Shock Proteins (HSPs) are involved in the repair of misfolded proteins and contribute to cellular homeostasis. We found that treatment with hibiscus mutabilis leaf extract significantly increased HSP-6:GFPfluorescence intensity, representing expression of HSP-6 protein in transgenic SJ4100 strain (J in FIG. 4). In addition, the cotton rose hibiscus leaf extract also inhibited the mortality of wild-type N2 nematodes fed with pseudomonas aeruginosa (PA14), indicating that cotton rose hibiscus leaf extract enhanced nematode resistance to pathogenic stress (H in fig. 4). In agreement, the cotton rose hibiscus leaf extract increased the expression of detoxification-associated protein GST-4 in transgenic CL2166 nematodes (fig. 4, K). In addition, the cotton rose hibiscus leaf extract also improved the resistance of nematodes to oxidative stress, which is reflected in the improved survival rate of N2 nematodes under oxidative stress induced by 20mM H2O2, and the up-regulated expression of SOD-3:: GFP protein in transgenic CF1553 nematodes (I and L in fig. 4). The results show that the cotton rose hibiscus leaf extract can improve the resistance of the nematodes to heat, pathogenic bacteria and oxidative stress.

3.7 Hibiscus mutabilis leaf extract exerts neuroprotective effects in model of AD, PD and HD nematodes by autophagy induction

Next, it was investigated whether the extract of leaves of cotton rose hibiscus exerts neuroprotective effects on nematodes by activating autophagy. Firstly, RNAi bacteria are utilized to inhibit the expression of 4 key autophagy-related genes bec-1, unc-51, vps-34 and lgg-1 in a nematode AD model, and then the paralysis rate of CL4176 nematodes and the deceleration rate of BR5270 nematodes are respectively determined. As shown in FIG. 5A and FIG. 6A, treatment with Hibiscus mutabilis leaf extract significantly reduced the paralysis rate of CL4176 insect fed with control bacterium HT 115. However, feeding RNAi bacteria successfully counteracted the ameliorating effect of extracts of cotton rose hibiscus leaves on abeta-toxic protein-induced paralysis in CL4176 nematodes. Similarly, when BR5270 nematodes were fed with RNAi bacteria of these 4 key autophagy-related genes, the improvement effect of hibiscus mutabilis leaf extract on Tau-induced food perception behavior deficiency was also reversed (B in fig. 5). These results indicate that extracts of leaves of Hibiscus mutabilis may exert therapeutic effects against AD on nematodes by activating autophagy. Then, we performed similar RNAi bacterial feeding experiments to study the association between the autophagy induced by Hibiscus mutabilis leaf extract and the anti-PD and anti-HD effects. The results show that the previous cotton rose hibiscus leaf extract reduced the accumulation of NL5901 nematode alpha-synuclein alpha protein and improved dyskinesia (C and D in fig. 5 and B in fig. 6), DA degeneration of BZ555 nematode (E in fig. 5 and C in fig. 6), and improvement of polyQ40 accumulation of AM141 nematode were all counteracted by down-regulating 4 key autophagy genes by RNAi bacteria (fig. 5F and 6D). The data indicate that the neuroprotective effects provided by cotton rose hibiscus leaf extract in models of AD, PD and HD nematodes all depend on inducing autophagy.

3.8 Hibiscus mutabilis leaf extract induces autophagy in stabilized GFP-RFP-LC 3U 87 cells and PC-12 cells

To confirm whether the extract of leaves of Hibiscus mutabilis induces autophagy in vitro, stable GFP-RFP-LC 3U 87 cells were used to detect autophagy activity after the treatment of the extract of leaves of Hibiscus mutabilis. In the part, the cytotoxicity of the cotton rose hibiscus leaf extract on RFP-LC 3U 87 cells is firstly detected by using a CCK-8 kit, no obvious cytotoxicity exists below 31.3 mu g/mL, and the cotton rose hibiscus leaf extract with 7.8 mu g/mL has strong proliferation effect (A in figure 7). Subsequently, we tested autophagy of less than 31.3 μ g/mL of Hibiscus mutabilis leaf extract in RFP-LC 3U 87 cells. As shown in B and C of FIG. 7, the dose-dependent increase of the ratio of the number of GFP-LC3 fluorescent spots was observed in the extract of Hibiscus mutabilis leaves, indicating that the extract of Hibiscus mutabilis can induce autophagy in cells. Subsequently, whether the cotton rose hibiscus leaf extract can induce autophagy of nerve cells is further determined. Similarly, the cytotoxicity of Hibiscus mutabilis leaf extract in PC-12 cells was first examined. The results show that the cotton rose leaf extract obviously increases the cell activity at 2.0-62.5 mu g/mL, and the cotton rose leaf extract with 7.8 mu g/mL also shows the highest cell activity (D in figure 7). Then, we tested the expression levels of p62, LC 3-I and LC 3-II proteins by Western blotting. The results show that the extract of leaves of Hibiscus mutabilis dose-dependently reduced the expression of p62 protein in PC-12 cells and increased the ratio of LC 3-II/LC 3-I (E-G in FIG. 7). The result shows that the cotton rose hibiscus leaf extract can induce autophagy of GFP-RFP-LC 3U 87 cells and PC-12 cells.

3.9 Hibiscus mutabilis leaf extract inhibits A beta fiber-induced cytotoxicity in PC-12 cells

In order to explore whether the cotton rose hibiscus leaf extract plays a neuroprotective role in vitro, the improvement effect of the cotton rose hibiscus leaf extract on AD-related diseases is researched by adopting PC-12 cells. The formation of amyloid fibrils of a β is a major neuropathological marker of AD. In this study, inhibition of Abelmoschus manihot leaf extract on A β fibril formation was examined by ThT fluorescence. As shown in A in figure 8, the fluorescence intensity of ThT is weakened in a dose-dependent manner in 24h, 48h and 72h by using the cotton rose hibiscus leaf extract with different concentrations, which shows that the cotton rose hibiscus leaf extract can inhibit the formation of Abeta (1-42) fibers. In addition, there is increasing evidence that abnormal accumulation of a β fibers leads to neuronal degeneration and death. In order to research whether the folium Hibisci Mutabilis extract can reduce the cytotoxicity of Abeta fibril on nerve cells, an Abeta fibril induction model is used for constructing a nerve cell injury model, and then the treatment effect of the folium Hibisci Mutabilis extract on the model is detected by using a CCK-8 and PI/Hoechst staining method. As shown in B of FIG. 8, A β (1-42) fibrils significantly decreased the cell survival of PC-12 cells, while the cotton rose leaf extract dose-dependently increased the cell survival at a concentration range of 2-8 μ g/mL after drying. In addition, PI/Hoechst staining showed that Hibiscus mutabilis leaf extract significantly reduced the number of PI-positive cells in A β (1-42) -induced PC-12 cells (C in FIG. 8). The result shows that the cotton rose hibiscus leaf extract can destroy the formation of the Abeta (1-42) fibers and reduce the cytotoxicity induced by the Abeta (1-42) fibers in cells.

3.10 Hibiscus mutabilis leaf extract inhibits the expression of APP, Tau and Tau-P301L proteins in PC-12 cells

It is well known that a β is produced by proteolytic cleavage of Amyloid Precursor Protein (APP) by β -and γ -secretases, and that up-regulation of APP promotes the pathogenesis of AD by promoting a β production. Therefore, we examined whether the extract of leaves of Hibiscus mutabilis could reduce the production of A β by inhibiting the expression of APP in vitro. First, fluorescence microscopy analysis showed that the extract of leaves of Hibiscus mutabilis did not affect the transfection effect of pEGFP-N1 in PC-12 cells. We then used the pEGFP-N1-APP plasmid transfection to transiently over-express APP protein in PC-12 cells. As shown in D in FIG. 8, PC-12 cells transfected with pEGFP-N1-APP exhibited higher GFP intensity than control cells, indicating that the APP protein was overexpressed in PC-12 cells. However, the intensity of GFP is obviously reduced after the treatment of the cotton rose hibiscus leaf extract, which shows that the cotton rose hibiscus leaf extract can inhibit the expression of APP in vitro. Furthermore, neurofibrillary tangles (NFTs) composed of hyperphosphorylated Tau are another major pathological hallmark of AD. Then, we investigated whether or not the extract of leaves of Hibiscus mutabilis inhibited the expression of Tau and Tau-P301L proteins using PC-12 cells transiently transfected with pRK5-EGFP or pRK 5-EGFP-Tau-P301L. As shown by E-F in FIG. 8, GFP intensity, which represents the expression level of Tau or Tau-P301L, was significantly decreased in PC-12 cells after treatment with a leaf extract of Hibiscus mutabilis. All data show that the extract of the leaves of the cotton rose hibiscus can inhibit the expression of APP, Tau and Tau-P301L protein in vitro.

3.11 Hibiscus mutabilis leaf extract promotes degradation of APP, Tau and Tau-P301L proteins by inducing autophagy in PC-12 cells

To investigate whether the cotton rose hibiscus leaf extract promotes the degradation of AD-associated proteins by activating intracellular autophagy, autophagy was blocked using autophagy inhibitors 3-MA and Baf, and then the expression of APP, Tau and Tau-P301L proteins was assessed by fluorescence microscopy analysis. We observed that the fluorescence intensity of PC-12 cells transfected with pEGFP-N1-APP, pRK5-EGFP-Tau or pRK5-EGFP-Tau-P301L plasmid was significantly reduced after treatment with Hibiscus mutabilis leaf extract, while autophagy inhibitors, including 3-MA and Baf, significantly counteracted the effect of Hibiscus mutabilis leaf extract on the degradation of AD-associated proteins in PC-12 cells. In addition, Western blotting with GFP antibody was further performed to examine the expression of APP, Tau and Tau-P301L proteins. The results show that 3-MA and Baf obviously inhibit the clearance effect of the cotton rose hibiscus leaf extract on APP, Tau and Tau-P301L proteins (G-I in figure 8). The result shows that the cottonrose hibiscus leaf extract promotes the degradation of AD-related proteins in vitro through autophagy induction.

3.12 Hibiscus mutabilis leaf extract improves cognitive and memory disorders in 3 × Tg-AD mice

A three transgenic 3 x Tg-AD model mouse expressing three mutant transgenes (i.e., PS1M146V knock-in, APPS we and tauP301L) is currently the most popular AD mouse model because it presents a β and Tau lesions, neuroinflammation, cognitive decline and memory impairment in an age-dependent manner. Here, the neuroprotective effect of Hibiscus mutabilis leaf extract on 6-month old 3 × Tg-AD mice was studied. Cognitive ability was first assessed in the study using the Y Maze (YM) test. As shown in A and B in FIG. 9, the number of times 3 × Tg-AD mice entered the new arm was significantly reduced compared to WT mice, indicating that the cognitive ability of 3 × Tg-AD mice was impaired. However, the percentage of 3 × Tg-AD mice that entered the new arm after treatment with cotton rose hibiscus leaf extract significantly increased in a dose-dependent manner. These data indicate that extracts of Hibiscus mutabilis leaves can improve spatial working memory in 3 × Tg-AD mice.

Next, the Morris Water Maze (MWM) test was used to further evaluate spatial learning and memory. During training, escape latencies, i.e. the time at which the mouse found the hidden platform, were recorded. It was observed that escape latency of WT mice decreased significantly in five consecutive days of training, with the value on day five (39.0%) being only 43.3% of the value on day one (90.0%). In contrast, the escape latency for the model group to find the hidden platform (83.8%) was still 93.1% of the first day (90.0%) and 2.15 times the WT group time, indicating that the learning ability of the 3 × Tg-AD mice was impaired (C and D in fig. 9). In contrast, the escape latency of the last training day was also dose-dependently decreased in 3 × Tg-AD mice treated with cotton rose hibiscus leaf extract at doses of 50, 100 and 200mg/kg, which were 45.8%, 27.1% and 17.1%, respectively (C and D in fig. 9). In addition, the mean time percentage in the target quadrant, the frequency of crossing the original platform quadrant and the mean distance percentage in the target quadrant were all decreased in 3 × Tg-AD mice compared to WT mice, which were successfully prevented by treatment with extracts of cotton rose hibiscus leaf (E-G in figure 9). The data show that the cotton rose hibiscus leaf extract improves the cognitive and memory disorders of 3 XTG-AD mice.

3.13 Hibiscus mutabilis leaf extract for alleviating Abeta and Tau disorders in 3 XTG-AD mice

A β plaques and intracellular NFTs are the major characteristic lesions of AD. In order to research whether the extract of cotton rose hibiscus leaves can improve the A beta and NFTs pathological changes of 3 XTg-AD mice, the expression of the A beta and p-Tau protein is detected by an immunohistochemical method. The results showed that 3 × Tg-AD mice had higher levels of A β deposition and p-Tau protein expression in hippocampus and cerebral cortex (A in FIG. 10) compared to WT mice, which is consistent with previous results. In contrast, significant reductions in Abeta deposition and p-Tau levels were observed in 3 XTG-AD mice treated with Hibiscus mutabilis leaf extract (A in FIG. 10). In addition, brain tissue and serum A β (1-42) levels were assessed by ELISA. The results showed that the brain tissue and serum A.beta. (1-42) content of 3 × Tg-AD mice was increased compared to WT mice, and that treatment with Hibiscus mutabilis leaf extract reversed this phenomenon significantly (B and C in FIG. 10). In addition, Western blotting showed that Hibiscus mutabilis leaf extract also prevented abnormal elevated expression levels of A β and p-tau proteins in the brains of 3 × Tg-AD mice (D-F in FIG. 10). These results suggest that treatment with extracts of cotton rose hibiscus leaves has a positive effect on improving abeta plaque formation and Tau phosphorylation in 3 x Tg-AD mice.

Previous reports have shown that accumulation of a β oligomers and p-Tau may be responsible for neurological damage. The present study examined neuronal health by HE and Nissl staining. HE staining results showed that in untreated 3 × Tg-AD mice, neurons in the hippocampus and cerebral cortex were largely lost, and many of the remaining neurons were shown to be basophilic, nuclear, deep-stained and shriveled. However, in the hippocampus (CA1-CA2), treatment with an extract of Hibiscus mutabilis leaves significantly increased the number of surviving neurons and decreased the number of basophilic neurons (H in FIG. 10). In addition, Nissl staining showed that the neurones Nissl body of hippocampus and cerebral cortex was significantly increased and volume was larger after treatment with the Hibiscus mutabilis leaf extract, indicating that the structure and function of neurones were improved after treatment with the Hibiscus mutabilis leaf extract (I in FIG. 10). In addition, in order to further clarify the intrinsic molecular mechanism of the lesion, a protein Bax promoting apoptosis and a protein Bcl-2 inhibiting apoptosis in brain tissue were detected by Western blotting. We observed a significant increase in Bax/Bcl-2 ratio in 3 XTG-AD mice, which was reversed after treatment with extracts of Hibiscus mutabilis leaves (D and G in FIG. 10). Taken together, these data indicate that Hibiscus mutabilis leaf extract can reduce the pathological changes in 3 × Tg-AD mice.

3.14 Hibiscus mutabilis leaf extract inhibits activation of NLRP3 inflammasome and induces activation of autophagy in 3 × Tg-AD mice

The NLRP3 inflammasome is an obvious inflammatory body and consists of a proteolytic complex formed by NLRP3, ASC and Caspase-1, leads to the generation of Caspase-1 mediated Interleukin (IL) -1 beta and IL-18 in microglia, and finally promotes the development and the progress of AD. In the research, the inhibition effect of the cotton rose hibiscus leaf extract on NLRP3 inflammasome of 3 XTg-AD mice is further researched. Western blotting results show that the expression of 5 key NLRP3 inflammation related proteins including NLRP3, Caspase-1, ASC, IL-1 beta and IL-18 is obviously reduced in 3 × Tg-AD mice (shown in a figure 11, A-F), and that the cotton rose hibiscus leaf extract inhibits NLRP3 inflammation bodies in vivo. Furthermore, it was found that extracts of leaves of Hibiscus mutabilis induced autophagy in 3 XTg-AD mice, as evidenced by a decrease in p62 protein expression and an increase in LC3B levels in brain tissue (A, G and H in FIG. 11), consistent with the observations of C.elegans and PC-12 cells. The result shows that the cotton rose hibiscus leaf extract can inhibit NLRP3 inflammasome and activate autophagy of 3 × Tg-AD mice.

4. Summary of the invention

(1) Extensive and preliminary screening using a transgenic C.elegans model revealed that extracts of Hibiscus mutabilis have strong autophagy-inducing activity, as evidenced by a decrease in the protein levels of SQST-1/p 62:GFPin BC12921 nematodes and an increase in the accumulation of GFP:LGG-1/LC 3 positive spots in DA2123 nematodes.

(2) The neuroprotective effect of the cotton rose leaf extract is determined by adopting a plurality of mature caenorhabditis elegans models, the cotton rose leaf extract treatment obviously promotes A beta degradation, delays paralysis, and improves food perception disorder of transgenic nematodes expressing human A beta or Tau protein through neurons or muscles, and the data indicate that the cotton rose leaf extract can effectively improve AD symptoms and has the neuroprotective property aiming at protein aggregation of the caenorhabditis elegans. This is shown by the reduced accumulation of alpha-synuclein and polyQ40 aggregates, improved dyskinesia, and recovery of neurons from nematodes expressing alpha-synuclein or polyQ40 after extraction with cotton rose hibiscus leaf extracts, which all demonstrated similar results in the PD and HD models of C.elegans. In conclusion, it was determined that the extract of leaves of Hibiscus mutabilis has a neuroprotective effect on aggregate-associated NDs.

(3) The treatment of the cotton rose hibiscus leaf extract can obviously prolong the life of wild type N2 nematodes, improve the physique and enhance the resistance to heat, pathogenic bacteria and oxidative stress, and the results show that the cotton rose hibiscus leaf extract is one of promising components in the medicaments for treating aging and aging-related diseases (such as AD, PD and HD).

(4) The most common AD in NDs was selected to study the neuroprotective efficacy of Hibiscus mutabilis leaf extract in other models, and by using PC-12 cells, it was found that treatment with Hibiscus mutabilis leaf extract significantly inhibited the cytotoxicity of Abeta fibers and APP, Tau and Tau P301L proteins in vitro. In addition, it was found that cognitive and memory functions as well as AD pathology in 3 × Tg-AD mice were also improved by administration of Hibiscus mutabilis leaf extract. Therefore, the neuroprotective effect of the cotton rose leaf extract is verified and confirmed in various cells and animal models, and the neuroprotective effect of the cotton rose leaf extract in other models is further verified, so that the feasibility of clinical application is improved.

(5) Misfolded proteins associated with NDs are degraded mainly by autophagy. The cotton rose hibiscus leaf extract can promote the formation of GFP-LC3 spots in stable GFP-RFP-LC 3U 87 cells, increase the expression of LC3B in PC-12 cells and 3 × Tg-AD mice and reduce the expression of autophagy substrate P62 protein, and further verifies the autophagy induction effect of the cotton rose hibiscus leaf extract in vitro and in vivo. In order to determine whether the autophagy induction is related to the neuroprotective effect of the cotton rose leaf extract in various models, 4 key autophagy related genes are knocked out in an AD nematode model by adopting RNAi bacteria, and the result shows that the neuroprotective effect of the cotton rose leaf extract is obviously counteracted. Furthermore, when autophagy inhibitors 3-MA and Baf blocked autophagy activity, the degradation of APP, Tau and Tau P301L proteins by extracts of Hibiscus mutabilis was reversed in PC-12 cells. In 3 × Tg-AD mice, extracts of leaves of Hibiscus mutabilis reduced cognitive and memory impairment and inhibited inflammatory responses and apoptosis associated with autophagy activation. The results show that the cotton rose hibiscus leaf extract has obvious treatment effect on NDs of various caenorhabditis elegans models, cells models and animal models through autophagy induction.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principles of the present invention are intended to be included within the scope of the present invention.

Claims (7)

1. The application of the cotton rose hibiscus leaf extract in the medicines for treating neurodegenerative diseases is characterized in that the cotton rose hibiscus leaf extract is a cotton rose hibiscus leaf ethanol extract.

2. The use of the cotton rose hibiscus leaf extract as claimed in claim 1, wherein the cotton rose hibiscus leaf extract is a 65-80 v% ethanol extract of cotton rose hibiscus leaves.

3. The use of the cotton rose hibiscus leaf extract as claimed in claim 1, wherein the dosage of the cotton rose hibiscus leaf extract is greater than or equal to 4.0 mg/kg.

4. The use of the HlblSClSClSClSCHI leaf extract in the medicament for treating neurodegenerative diseases according to claim 3, wherein the dose of the HlblSCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCLE leaf extract is 4.0-25 mg/kg.

5. The use of the HlblSClSClSClSCHI leaf extract in the medicament for treating neurodegenerative diseases according to claim 4, wherein the dose of the HlblSCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCLE leaf extract is 4.0-16.3 mg/kg.

6. The use of the HlblSClSClSClSCHI leaf extract in the medicament for treating neurodegenerative diseases according to claim 5, wherein the dose of the HlblSCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCHISCLE leaf extract is 8-16.3 mg/kg.

7. The use of the extract of leaves of Hibiscus mutabilis according to any one of claims 1 to 6 in the preparation of a medicament for treating neurodegenerative disease, wherein the ethanol extract of leaves of Hibiscus mutabilis is prepared by:

soaking folium Hibisci Mutabilis in ethanol, and performing ultrasonic treatment; removing ethanol in the soaking solution after ultrasonic extraction, and then performing freeze-drying to obtain dry powder, namely the ethanol extract of the leaves of the cotton rose.

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