阅读说明：本技术 小檗胺在制备治疗阿尔兹海默症药物中的应用 (Application of berbamine in preparation of medicine for treating Alzheimer's disease ) 是由 陈长兰 王海利 钱峰 吕晶 于 2021-11-08 设计创作，主要内容包括：本发明公开了小檗胺在制备治疗阿尔兹海默症药物中的应用。本发明具体涉及盐酸小檗胺在阿尔兹海默症治疗中的应用,表明了盐酸小檗胺显著地改善阿尔茨海默症小鼠的病理特征,提高阿尔茨海默症小鼠的学习记忆能力,对阿尔茨海默症具有一定的治疗作用。(The invention discloses application of berbamine in preparing a medicament for treating Alzheimer's disease. The invention particularly relates to application of berbamine hydrochloride in treating Alzheimer's disease, which shows that the berbamine hydrochloride can remarkably improve the pathological characteristics of Alzheimer's disease mice, improve the learning and memory abilities of the Alzheimer's disease mice, and has a certain treatment effect on the Alzheimer's disease.)

1. Application of berbamine in preparing medicine for treating Alzheimer disease is provided.

2. Use according to claim 1, characterized in that: application of berbamine in preparing a medicament for reducing formation of Abeta plaque in hippocampal tissue of brain of a patient with Alzheimer's disease.

3. Use according to claim 1, characterized in that: application of berbamine in preparing medicine for reducing neurofibrillary tangle formation in hippocampal tissue of brain of patient with Alzheimer's disease.

4. Use according to claim 1, characterized in that: application of berbamine in preparing medicine for reducing nerve cell pathological changes of patients with Alzheimer's disease.

5. Use according to claim 1, characterized in that: application of berbamine in preparing medicine for inhibiting calpain expression in brain tissue of Alzheimer disease patient.

6. Use according to claim 1, characterized in that: application of berbamine in preparing a medicament for promoting expression of selenoprotein K in brain tissues of patients with Alzheimer's disease.

Technical Field

The invention belongs to the field of medicines, and particularly relates to an application of berbamine in preparation of a medicine for treating Alzheimer's disease.

Background

Alzheimer's Disease (AD), the most common dementia in the world, affects as many as 3500 million people worldwide. Clinically, it is more common in the elderly, and is mainly characterized by patients showing different degrees of decline in memory and cognitive abilities. At present, the aging problem of the population of China is more and more serious, and the occurrence of AD seriously threatens the physical and psychological health of the old. Therefore, the prevention and treatment of AD has attracted scientific attention. Currently, there are only a limited number of drugs clinically used to alleviate the development of AD.

With the progress of research, the pathogenesis of AD is becoming more and more clear, and early theories of the development of alzheimer's disease have been increasingly confirmed. The method mainly comprises the following steps: the oxidative stress hypothesis that excessive cellular oxidation causes damage to neurons; the amyloid beta polypeptide cascade hypothesis that a large amount of a β produced and accumulated may lead to a complex pathological cascade, ultimately leading to the occurrence of neurodegenerative diseases such as alzheimer's disease; the Tau protein hypothesis that excessive A beta may cause hyperphosphorylation of Tau protein, and finally cause reduction of the binding capacity of Tau protein and microtubules to cause instability of cytoskeleton, thereby forming neurofibrillary tangles and causing damage and death of nerve cells; glial cells release inflammatory factors such as interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), transforming growth factor-beta (TGF-beta), etc., which further activate the non-activated glial cells, make the inflammatory response more severe and damage neurons, and also make A beta aggregate to further expand the inflammatory response hypothesis, neurovascular hypothesis, metabolic hypothesis, etc. of the inflammatory response. Mutations at the gene level and other causes of infectivity, damage to the blood brain barrier, etc., can all cause diseases.

Because AD is not curable before, treatment tends to alleviate the symptoms of AD as well as reduce degenerative neuropathy and cell death. The level of acetylcholine in the brain is increased by acetylcholinesterase inhibitors. Some treatments aim to reduce the time to invasion of brain function. Non-steroidal anti-inflammatory drugs can reduce the neuroinflammatory response caused by AD. Antiviral treatment may reduce viral infections associated with AD mainly caused by herpes virus, hepatitis c virus, and the like. Pyramidal cytopathic effects, hyperactivated microglia and microglial accumulation are also important pathological features of AD.

The berbamine has a structural formula shown in (I), and the liensinine is a bisbenzylisoquinoline alkaloid which is a natural product widely existing in berberis plants and has various pharmacological activities. In addition, berbamine hydrochloride is used as a mature preparation on the market and has been widely applied clinically, but the application of berbamine in treating Alzheimer disease is not reported yet.

Disclosure of Invention

Based on the pathogenesis and relevant pathological characteristics, the invention aims to preliminarily discuss whether the berbamine hydrochloride can inhibit the expression of calpain as the liensinine, so as to improve the expression of the selenoprotein SelK and play a certain role in resisting Alzheimer disease, thereby providing a new theoretical basis for the drug treatment and prevention of AD and benefiting more AD patients.

The invention provides an application of berbamine in preparing a medicament for treating Alzheimer's disease.

Further, the invention provides an application of the berbamine in preparing a medicament for reducing the formation of A beta plaques in the brain hippocampal tissues of patients with Alzheimer's disease.

Further, the invention provides an application of the berbamine in preparing a medicament for reducing the formation of neurofibrillary tangles in the hippocampal tissues of the brain of a patient with Alzheimer's disease.

Further, the invention provides an application of the berbamine in preparing a medicine for reducing nerve cell pathological changes of patients with Alzheimer's disease.

Further, the invention provides application of the berbamine in preparing a medicament for inhibiting calpain expression in brain tissues of patients with Alzheimer's disease.

Further, the invention provides application of the berbamine in preparing a medicament for promoting expression of selenoprotein K in brain tissues of patients with Alzheimer's disease.

The invention has the beneficial effects that: berbamine as a medicine on the market belongs to tertiary amine alkali, is alkalescent, has good intestinal absorption, has the advantages of high safety and low toxic and side effect, and forms a good stable dosage form. According to the invention, three AD model mice, namely a brain-located injection aluminum trichloride AD mouse, an intraperitoneal injection D-galactose and perfusion aluminum trichloride AD mouse and an APP/PS1 double-transgenic AD mouse are subjected to hydrochloric acid berbamine perfusion for 40 days in a gastric perfusion administration mode, physiological and biochemical indexes of learning and memory capacity, A beta plaque formation, neuron fiber tangle formation, nerve cell pathological changes and the like of each group of mice are measured, a part of brain tissues are taken to prepare paraffin tissue sections, and tissue change research is carried out through immunohistochemical staining, glycine silver plating staining and hematoxylin-eosin staining, and the influence of the hydrochloric acid berbamine on calpain and SelK expression in the three Alzheimer disease model mice is researched through Western blot. Research shows that the berbamine can obviously reduce or improve relevant pathological indexes of the Alzheimer disease. The invention provides a new medicinal application of the berbamine, shortens the research and development time of new medicaments for treating the Alzheimer disease, and saves the research and development cost of related new medicaments.

Drawings

FIG. 1-1 shows the construction of a mouse model of brain-specific injection of aluminum trichloride AD.

FIG. 1-2 shows the effect of berbamine hydrochloride administration on escape latency in brain-localized injection of aluminum trichloride AD mouse model mice.

FIGS. 1-3 show the effect of berbamine hydrochloride administration on the time around the station of mouse model mouse with brain-specific injection of aluminum trichloride AD.

FIGS. 1-4 show the effect of berbamine hydrochloride administration on the number of station crossings of a brain-specific injection of aluminum trichloride AD mouse model mouse.

FIGS. 1-5 show the effect of berbamine hydrochloride administration on the formation of A β plaques in a mouse model of brain-localized injection of aluminum trichloride AD (immunohistochemical staining, 200X).

FIGS. 1-6 show the effect of berbamine hydrochloride administration on brain-specific injection of aluminum trichloride AD mouse model mouse hippocampal pyramidal cells (HE staining results, 400X).

FIGS. 1-7 are graphs showing the effect of berbamine hydrochloride administration on neurofibrillary tangle formation in a mouse model mouse with localized injection of aluminum trichloride AD (silver plating glycine staining results, 400X).

FIGS. 1 to 8 show the effect of berbamine hydrochloride administration on the expression of Calpain protein in the brain of AD mouse model mice injected with aluminum trichloride;

wherein, a is an electrophoresis picture of Calpain protein, and b is a histogram of Calpain protein expression.

FIGS. 1 to 9 show the effect of berbamine hydrochloride administration on the expression of SelK protein in the brain of AD mouse model mice injected with aluminum trichloride in a localized manner;

wherein, a is a SelK protein electrophoresis chart, and b is a histogram of SelK protein expression level.

FIG. 2-1 is a graph showing the effect of berbamine hydrochloride administration on the escape latency of intraperitoneal injection of D-galactose and gavage aluminum trichloride AD mouse model.

FIG. 2-2 is a graph showing the effect of berbamine hydrochloride administration on the time around the station of intraperitoneal injection of D-galactose and gavage of aluminum trichloride AD mouse models.

FIG. 2-3 shows the effect of berbamine hydrochloride administration on the number of times that mice model AD mice injected with D-galactose and gavage aluminum trichloride cross the station.

FIGS. 2-4 show the effect of berbamine hydrochloride administration on Abdominal D-galactose and gavage aluminum trichloride AD mouse model mouse Abeta plaque formation (immunohistochemical staining results, 200X).

FIGS. 2-5 show the effect of berbamine hydrochloride administration on D-galactose and gavage aluminum trichloride AD mouse model mouse hippocampal pyramidal cells (HE staining results, 400X).

FIGS. 2-6 are graphs showing the effect of berbamine hydrochloride administration on the formation of neurofibrillary tangles in intraperitoneal D-galactose and gavage aluminum trichloride AD mouse model (silver plating glycine staining results, 400X).

FIGS. 2-7 show the effect of berbamine hydrochloride administration on the expression of Calpain protein in the brain of an intraperitoneal injection D-galactose and gavage aluminum trichloride AD mouse model;

wherein, a is an electrophoresis picture of Calpain protein, and b is a histogram of Calpain protein expression. .

FIGS. 2 to 8 show the effect of administration of berbamine hydrochloride on the expression of SelK protein in the brain of an intraperitoneal injection of D-galactose and gavage aluminum trichloride AD mouse model;

wherein, a is an electrophoresis chart of the SelK protein, and b is a bar chart of the SelK protein expression level. .

FIG. 3-1 is a graph of the effect of berbamine hydrochloride administration on escape latency in APP/PS1 dual transgenic AD mouse model mice.

FIG. 3-2 is a graph showing the effect of berbamine hydrochloride administration on the time around the platform of APP/PS1 dual transgenic AD mouse model.

FIG. 3-3 is a graph showing the effect of berbamine hydrochloride administration on the number of platform crossings in APP/PS1 dual transgenic AD mouse model mice.

FIGS. 3-4 are graphs showing the effect of berbamine hydrochloride administration on the formation of plaques in the APP/PS1 dual transgenic AD mouse model mouse A β (immunohistochemical staining, 200X).

FIGS. 3-5 are graphs showing the effect of berbamine hydrochloride administration on hippocampal pyramidal cells of APP/PS1 dual transgenic AD mouse model mice (HE staining results, 400X).

FIGS. 3-6 are graphs showing the effect of berbamine hydrochloride administration on neurofibrillary tangle formation in a mouse model of APP/PS1 dual transgenic AD (silver plating glycine staining results, 400X).

FIGS. 3-7 show the effect of berbamine hydrochloride administration on Calpain protein expression in brain of APP/PS1 dual transgenic AD mouse model;

wherein, a is an electrophoresis picture of Calpain protein, and b is a histogram of Calpain protein expression.

FIGS. 3-8 Effect of berbamine hydrochloride administration on SelK protein expression in brain of mice model APP/PS1 dual transgenic AD mice;

wherein, a is an electrophoresis chart of the SelK protein, and b is a bar chart of the SelK protein expression level.

Detailed Description

To further illustrate the present invention, a series of examples are given below, which are purely illustrative and are intended to be a detailed description of the invention only and should not be understood as limiting the invention.

Example application of Berberine in preparation of medicine for treating Alzheimer's disease

The method comprises the following steps: the qualified mice are randomly divided into a brain-localization injection aluminum trichloride AD mouse model group, an intraperitoneal injection D-galactose and gavage aluminum trichloride AD mouse model group, an APP/PS1 double-transgene AD mouse model group, a control group, a pseudo-operation group (the pseudo-operation group is used for controlling the brain-localization injection group, only the same operation of scratching the scalp is carried out, and the positioning injection is not carried out) and a berbamine hydrochloride administration group. The administration group is administrated to the berbamine hydrochloride for intragastric administration for 40 days, and after 40 days, whether the berbamine hydrochloride can play a certain role in improving the learning and memory abilities of the model mice is researched through a water maze experiment. Meanwhile, the effects of the berbamine hydrochloride on the formation of Abeta plaques, the formation of neurofibrillary tangles, the pathological changes of nerve cells and the like in the hippocampal tissues of the mouse model of the Alzheimer disease are researched by carrying out experiments such as immunohistochemical staining, glycine silvering staining, hematoxylin-eosin staining (HE staining) and the like on paraffin sections of the brain tissues of the mouse. And the influence of the berbamine hydrochloride on the expression of calpain and SelK in 3 Alzheimer model mice is researched by western blot.

Research on therapeutic effect of berbamine hydrochloride on brain-targeted injection of aluminum trichloride AD mice

Establishing an Alzheimer's disease mouse model: after anesthetizing the mice, the hair of the head was removed, the scalp was cut open and the membrane was cut open until the skull was exposed intact, as shown in fig. 1-1. The mouse is fixed on a frame of a brain positioning instrument, and a sample injection needle is filled with an aluminum trichloride aqueous solution with the concentration of 2.5 percent. The anterior skull of a mouse is taken as a coordinate origin, the posterior part of the anterior skull is 3.25mm, the lateral opening is 2.62mm, the part below the skull is 3mm and is taken as a coordinate point, a dental drill is used for drilling a small hole on the skull at the coordinate point, a micro-injection pump is used for slowly injecting 3 mu L of the injection at the speed of 1 mu L/min, and no aluminum trichloride aqueous solution seeps out at the position of the brain in the injection process.

As can be seen from fig. 1-2, the mice underwent the water maze (escape latency training) experiment for four days, the escape latencies of the control group and the sham operation group were significantly reduced, while the escape latencies of the mice of the AD mouse model group injected with alchlor in brain localization were substantially unchanged, and the difference between the escape latencies of the model group and the sham operation group was significantly increased from the second day of training. The escape latency of the mice in the berbamine hydrochloride administration group is obviously shortened compared with that in the model group, is close to that in the control group and the sham operation group, and is most obvious from the third day to the fourth day. The result shows that the spatial memory ability of the mice is obviously improved after the administration of the berbamine hydrochloride for 40 days.

As can be seen from FIGS. 1 to 3, on the fifth day after training, the peripheral movement time of the mouse platform of the AD mouse model group injected with aluminum trichloride in a brain-oriented manner was significantly shorter than that of the control group and the sham operation group, while the peripheral movement time of the mouse platform after administration of berbamine hydrochloride was significantly increased. As can be seen from FIGS. 1 to 4, on the fifth day after training, the number of times of crossing the platform by the mice of the AD mouse model group injected with the aluminum trichloride in the brain location was significantly lower than that of the control group and the sham operation group, while the number of times of crossing the platform by the mice of the berbamine hydrochloride administration group was significantly increased. The result shows that the learning and memory ability of the mice is obviously enhanced after the administration of the berbamine hydrochloride.

As can be seen from FIGS. 1 to 5, the results of immunohistochemical staining of Abeta showed that no significant Abeta plaque deposition was observed in the hippocampal tissues of the mice in the control group, the sham operation group and the administration group, while Abeta plaques appeared in the hippocampal tissues of the mice in the model group, indicating that the Berberine hydrochloride can inhibit the generation of Abeta plaques in the hippocampal tissues of the brains of AD mice.

As can be seen from FIGS. 1-6, the hippocampal pyramidal cells of the mice in the control group and the sham-operated group have complete morphology, are arranged regularly and densely, have large and round cell nuclei, obvious nuclear cytoplasm, clear cell boundary and no obvious abnormality. And the hippocampal pyramidal cells of the model group mice have irregular shapes, and the nucleus pulposus deep staining boundary is fuzzy, thereby presenting obvious pathological characteristics. The hippocampal pyramidal cells of the mice in the administration group have good cell state, regular shape, large and round cell nucleus and clear cytoplasm boundary. Therefore, the berbamine hydrochloride protects the hippocampal pyramidal nerve cells to a certain extent.

As can be seen from FIGS. 1 to 7, the hippocampal tissue of the control and sham-operated mice showed no neurofibrillary tangles, while the hippocampal tissue of the model mice showed dark brown neurofibrillary tangles, and the brain tissue of the mice administered with berbamine hydrochloride also showed no neurofibrillary tangles. The berbamine hydrochloride can inhibit the generation of neurofibrillary tangles.

As can be seen from fig. 1-8 and fig. 1-9, the Calpain expression level in the brain tissue of the mice in the control group and the sham-operated group is low, the selenoprotein K content is high, while the Calpain expression level in the brain tissue of the mice in the model group is high, the selenoprotein K content is low, and the expression level of the selenoprotein K in the brain tissue of the mice in the berbamine hydrochloride administration group is obviously higher than that of the mice in the model group and is obviously lower than that of the mice in the model group although the Calpain expression level is not high in the control group and the sham-operated group. Therefore, the berbamine hydrochloride can inhibit the expression of Calpain, thereby improving the expression of selenoprotein K.

Therapeutic action of berbamine hydrochloride on D-galactose and gavage aluminium trichloride AD mouse by intraperitoneal injection

As can be seen from FIG. 2-1, after the mice are subjected to a four-day water maze (escape latency training) experiment, although the mice in the model group and the mice in the control group are both reduced, the reduction degree of the mice in the model group is obviously slower than that of the mice in the control group, and a trend which is gentle is seen from the rear end of the curve, which indicates that the space memory capacity of the mice is damaged by the combined induction of aluminum chloride and D-galactose, the escape latency of the mice in the administration group is obviously shortened compared with that of the model group, and the improvement effect of the space memory capacity of the mice by the berbamine hydrochloride is approximately demonstrated by the control group.

As can be seen from FIGS. 2-2, on the fifth day after training, the activity time around the platform of the AD mouse model group injected with D-galactose and gavage aluminum trichloride was significantly shorter than that of the control group, while the activity time around the platform of the AD mouse model group administered with berbamine hydrochloride was significantly increased. As can be seen from FIGS. 2-3, on the fifth day after training, the number of times that the mouse injected with the AD mouse model group of aluminum trichloride in brain-localization mode crosses the platform is significantly lower than that of the control group, while the number of times that the mouse injected with berbamine hydrochloride is crossed the platform is significantly increased, which indicates that the learning and memory ability of the mouse is significantly enhanced after the administration of berbamine hydrochloride.

As can be seen from FIGS. 2-4, the A beta plaques are not seen in the hippocampal tissues of the control mice, the A beta plaques are seen in the hippocampal tissues of the model mice, and the A beta plaque deposition is not seen in the hippocampal tissues of the brain of the mice of the administration group, which indicates that the berbamine hydrochloride can inhibit the generation of the A beta plaques in the hippocampal tissues of the brain of the AD mice.

As can be seen from FIGS. 2-5, the hippocampal tissue of the control mice had intact cell morphology, aligned and dense arrangement, large and round cell nucleus, abundant cytoplasm and no pathological features. The hippocampal tissue cells of the model group mice have more pathological changes, irregular cell boundary and deeply-stained and solid-shrunk cell nucleuses. However, the cells of the berbamine hydrochloride administration group mice only have few pyramidal nerve cell nuclei to be condensed, and the cells are irregular in shape and rare in cytoplasm. Therefore, the berbamine hydrochloride can relieve the neuropathy of the hippocampal tissues of the AD mice.

As can be seen from FIGS. 2 to 6, there was no significant abnormality in the hippocampal tissue of the control group, while more brown neurofibrillary tangles appeared in the hippocampal tissue of the model group, and neurofibrillary tangles were not seen in the hippocampal tissue of the berbamine hydrochloride administration group. The berbamine hydrochloride is proved to have the function of inhibiting the formation of neurofibrillary tangles.

As can be seen from FIGS. 2-7 and FIGS. 2-8, the expression level of selenoprotein K in brain tissue of control mice is significantly higher than that of model mice, while the expression level of Calpain Calpain is significantly lower than that of model mice, the expression level of Calpain in brain tissue of mice administered with berbamine hydrochloride is lower than that of model mice, and the expression level of selenoprotein K is significantly higher than that of model mice though lower than that of control mice. Therefore, the berbamine hydrochloride can inhibit the expression of Calpain, thereby improving the expression of selenoprotein K.

Therapeutic effect of (tri) berbamine hydrochloride on APP/PS1 double-transgene AD mice

As can be seen from FIG. 3-1, after the mice are subjected to the water maze (escape latency training) experiment for four days, the escape latencies of the control group mice and the administration group mice are obviously reduced, while the reduction degree of the escape latencies of the model group mice after the training is not obvious and is lower than that of the other two groups. The result shows that the space memory ability of APP/PS1 mice is greatly improved after berbamine hydrochloride administration.

As can be seen from FIGS. 3-2, the activity time around the platform was significantly reduced in mice of APP/PS1 model group and significantly increased in mice after administration, after learning training for four days. As can be seen from FIGS. 3-3, after learning training, the APP/PS1 model group crossed the station significantly less than the control group, while APP/PS1 mice crossed the station significantly more after administration. Therefore, the berbamine hydrochloride has a great effect of improving the spatial learning and memory ability of the APP/PS1 double-transgenic mouse.

3-4, the hippocampal tissue of the control group mouse has no A beta plaque, while the hippocampal tissue of the APP/PS1 model group mouse has no A beta plaque deposition, and the brain hippocampal tissue of the administration group mouse has no A beta plaque deposition, which shows that the berbamine hydrochloride can inhibit the generation of the A beta plaque in the brain hippocampal tissue of the AD mouse.

As can be seen from FIGS. 3-5, the hippocampal tissue cells of the control group mice are complete in morphology, arranged regularly and densely, large and round in cell nucleus, rich in cytoplasm and free from pathological changes, and the hippocampal tissue cells of the APP/PS1 model group mice have more pathological changes, irregular cell division and deeply-stained and condensed cell nucleus. The hippocampal tissue cells of the berbamine hydrochloride administration group mice only have few pyramidal nerve cell nuclei to be condensed, and the cells are irregular in shape and rare in cytoplasm. The berberis alcaine can reduce neuropathy of hippocampal tissue of AD mice.

As can be seen from FIGS. 3-6, there was no significant abnormality in the hippocampal nerve fibers of the control mice, whereas more brown neurofibrillary tangles were present in the hippocampal tissue of the APP/PS1 model mice. Neurofibrillary tangles were not observed in the hippocampal tissues of the mice in the berbamine hydrochloride administration group. The berbamine hydrochloride is proved to have the function of inhibiting the formation of neurofibrillary tangles.

As can be seen from FIGS. 3-7 and 3-8, the expression level of selenoprotein K in brain tissue of control mice is significantly higher than that of model mice, the expression level of Calpain is significantly lower than that of model mice, the expression level of Calpain in brain tissue of mice administered with berbamine hydrochloride is lower than that of model mice, and the expression level of selenoprotein K is significantly higher than that of model mice though lower than that of control mice. Therefore, the berbamine hydrochloride can inhibit the expression of Calpain, thereby improving the expression of selenoprotein K.

In conclusion, by injecting a mouse model of inducing alzheimer disease into the brain of aluminum chloride, combining aluminum chloride with a mouse model of inducing alzheimer disease by D-galactose and a mouse model of APP/PS1 double-transgenic gene, observing the mouse behavior after berbamine hydrochloride administration, observing the staining results of slices of the mouse brain neurofibrillary tangles, abeta plaque deposits and the like, and by western blot experiment on brain tissues, the following conclusions can be drawn:

(1) the conclusion can be drawn by the water maze experiment: the berbamine hydrochloride has certain improvement effect on the space learning and memory ability of the Alzheimer disease model mouse induced by the three methods.

(2) As can be seen from the staining experiment of the paraffin section of the mouse brain tissue, the berbamine hydrochloride has the functions of reducing A beta and neurofibrillary tangles in the mouse hippocampal tissue and protecting hippocampal pyramidal cells.

(3) According to the results of Western blot experiments, analysis shows that the berbamine hydrochloride can reduce the degradation of the selenoprotein K by the calpain by inhibiting the expression of the calpain, so as to play a role in resisting Alzheimer's disease.