CN112451537A

CN112451537A - Application of baicalin in preparation of medicine for preventing and/or treating asymptomatic hyperuricemia and/or uric acid nephropathy

Info

Publication number: CN112451537A
Application number: CN202011281607.3A
Authority: CN
Inventors: 徐凌云; 邱银生; 刘永杰; 向慧龙
Original assignee: Wuhan Polytechnic University
Current assignee: Wuhan Polytechnic University
Priority date: 2020-11-16
Filing date: 2020-11-16
Publication date: 2021-03-09

Abstract

The invention belongs to the field of biological medicine, and relates to application of baicalin in preparation of a medicine for preventing and/or treating asymptomatic hyperuricemia and/or uric acid nephropathy. The invention researches the effect of baicalin on hyperuricemia nephropathy based on animal and cell experiments, and the result shows that the baicalin can inhibit the activity of xanthine oxidase, inhibit the generation of uric acid, reduce the serum uric acid level of a uric acid nephropathy mouse and obviously improve the renal function of the mouse; the baicalin can inhibit the damage of sodium urate crystals to rat renal tubular epithelial cells by reducing the secretion of TNF-alpha, IL-1 beta, lactate dehydrogenase and NO.

Description

Application of baicalin in preparation of medicine for preventing and/or treating asymptomatic hyperuricemia and/or uric acid nephropathy

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to application of baicalin in preparation of a medicine for preventing and/or treating asymptomatic hyperuricemia and/or uric acid nephropathy.

Background

In recent years, the incidence of asymptomatic hyperuricemia and uric acid nephropathy has increased significantly with the increase in living standard and the change in dietary structure. Approximately 2/3 uric acid produced by the human body is excreted with urine through the kidney, and therefore, hyperuricemia may cause a decrease in renal function. Uric acid nephropathy means that a large amount of urate crystals are deposited in a kidney collecting tube, a renal pelvis and a ureter in a short time and block the renal tubules, so that the proximal end of the renal tubules is expanded, acute renal function and severe injury are caused, acute renal failure is caused, or long-term high serum uric acid stimulates the kidney, so that kidney inflammation, fibrosis and other injuries are caused. At present, the drugs for treating the uric acid nephropathy are mainly based on chemical drugs, and mainly comprise xanthine oxidase inhibitors, such as: allopurinol, febuxostat, and the like; or drugs that promote uric acid excretion, such as: probenecid, benzbromarone, and the like. However, the above drugs have serious side effects such as: renal toxicity, anaphylaxis, cardiovascular toxicity, hepatorenal toxicity and the like, and the clinical use is severely limited. As can be seen, the traditional therapeutic drugs have great toxic and side effects and poor patient compliance.

In recent years, the traditional Chinese medicine has low toxic and side effects, definite curative effect and strong superiority, so that the traditional Chinese medicine is widely regarded for treating the uric acid nephropathy.

Disclosure of Invention

The invention aims to solve the problems that the existing drug for treating uric acid nephrosis has large toxic and side effects, is not easy to be tolerated by patients and is limited in clinical use. Provides a natural product-flavonoid compound with small toxic and side effects for treating hyperuricemia nephropathy. The protective effect of flavonoid quercetin on uric acid renal damage has been reported in literature. The invention firstly provides that the flavonoid compound baicalin can treat uric acid nephropathy, and the effect of the baicalin on resisting hyperuricemia nephropathy is researched from the animal and cell level. The invention aims to solve the problem of developing a high-efficiency and low-toxicity medicine for resisting hyperuricemia nephropathy, so as to solve the toxicity problem of the medicine used clinically, and improve the tolerance and the compliance of patients.

The first aspect of the present invention provides the use of baicalin in the preparation of a medicament for the prevention and/or treatment of asymptomatic hyperuricemia and/or uric acid nephropathy.

Baicalin is a light yellow powder with molecular formula C₂₁H₁₈O₁₁The molecular weight is 446.37, which is a flavone compound extracted and separated from root of Chinese medicinal material scutellaria root, and has wide pharmacological activity, such as antioxidant, antiphlogistic, antitumor, antibacterial and antiviral activities, and low toxicity. The baicalin is used for treating uric acid nephropathy, can safely and effectively play roles in reducing uric acid and protecting renal functions, and provides experimental basis for researching and developing high-efficiency low-toxicity natural medicines for treating uric acid nephropathy.

Wherein baicalin can be used alone or combined with allopurinol to form a compound, can reduce serum uric acid and renal oxidative stress level within the dosage range of 20-200mg/kg/d, has the effect of protecting renal function, and can be used for treating hyperuricemic nephropathy. Therefore, allopurinol can also be contained in the medicine.

According to the present invention, the uric acid nephropathy may include acute uric acid nephropathy, chronic uric acid nephropathy, and uric acid nephrolithiasis.

The baicalin can be orally administered or administered by injection, so that the medicament is in an oral dosage form or an injection dosage form. Experiments prove that 50mg/kg/d of baicalin is injected into the abdominal cavity, the hematuria level of uric acid nephropathy mice can be obviously reduced, the renal function can be protected, and the effect is better than that of the intragastric administration group with the same dose.

According to the invention, a mouse model of hyperuricemia nephropathy is prepared by an in vivo experimental method, and baicalin with the weight of 25mg/kg/d, 50mg/kg/d and 100mg/kg/d is adopted, and the result shows that the baicalin can obviously reduce the serum uric acid, urea nitrogen and creatinine levels of a uric acid nephropathy mouse, so that the application of the baicalin in preparing the medicine for reducing the serum uric acid, urea nitrogen and creatinine levels is provided in the second aspect of the invention. Baicalin can inhibit the activity of xanthine oxidase in mouse liver, thereby reducing uric acid production; the baicalin can remarkably reduce the kidney coefficient of a hyperuricemia kidney injury mouse to be close to the level of a normal mouse. In conclusion, the baicalin can inhibit the activity of xanthine oxidase, inhibit the generation of uric acid, reduce the serum uric acid level of a hyperuricemia nephropathy mouse and obviously improve the kidney function of the mouse.

According to the invention, a sodium urate crystal induced renal tubular epithelial cell injury model is established by using rat renal tubular epithelial cells (NKR-52e) as a research object through a cell experiment method, and baicalin with the concentration of 20 mu mol/L, 40 mu mol/L and 80 mu mol/L is used for intervention, and the result shows that the baicalin can remarkably reduce the expression of TNF-alpha, IL-1 beta, NO and lactate dehydrogenase in the supernatant of the model cells. In conclusion, the baicalin can inhibit the damage of sodium urate crystals to rat renal tubular epithelial cells by reducing the secretion of TNF-alpha, IL-1 beta, lactate dehydrogenase and NO. Accordingly, a third aspect of the present invention provides the use of baicalin in the preparation of TNF- α, IL-1 β, NO and lactate dehydrogenase inhibitors.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.

Figure 1 shows the level of LDH release (n-3) in cell culture supernatants, where, compared to the blank,^**P<0.01; in comparison to the set of models,^#P<0.05，^##P<0.01。

figure 2 shows the NO release levels (n-3) in cell culture supernatants, where, compared to the blank,^***P<0.001; in comparison to the set of models,^#P<0.05。

figure 3 shows the TNF-alpha expression levels (n-3) in cell culture supernatants, where, compared to the blank,^**P<0.01; in comparison to the set of models,^#P<0.05，^##P<0.01。

FIG. 4 shows a cell culture supernatant IL-1 beta tableTo a level (n-3), where, compared to the blank group,^**P<0.01; in comparison to the set of models,^#P<0.05，^##P<0.01。

Detailed Description

Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.

Examples

The following examples were studied using animal and cell experiments.

1. Research on effect of baicalin on uric acid nephropathy of mice

1.1 materials

1.1.1 Experimental animals

SPF male Kunming mouse, weight 20 ~ 22g, Sibeifu (Beijing) biotechnology limited company provides, animal pass certificate: SCXK (Jing) 20119-.

1.1.2 drugs and reagents

Baicalin (purity > 90%), batch number: C031B150902, available from kawa cooperative pharmaceutical limited; allopurinol (purity 98%), batch number: Y27O8C46913, available from shanghai source leaf biotechnology ltd; potassium oxonate (purity > 98%), batch number: p137112, available from Shanghai Allantin Biotechnology Ltd; uric acid assay kit (uricase colorimetric method), lot number: 20191129, respectively; urea nitrogen kit (urease method), batch No.: 20191211, respectively; creatinine kit (creatinine oxidase method), lot number: 20191218, respectively; coomassie brilliant blue kit, lot number: 20191217, respectively; xanthine Oxidase (XOD) kit, lot No.: 20191218, respectively; are purchased from Nanjing to build a bioengineering institute; yeast extract, batch number: 20180902, available from Obo Star Biotechnology, Inc., Beijing; sodium carboxymethylcellulose, dimethyl sulfoxide, available from national pharmaceutical group chemical reagents, ltd; double distilled water was prepared for the laboratory.

1.1.3 Experimental instruments

AL204 electronic analytical balance (mettler-toledo corporation); SPS2001F electronic balance (aohaus corporation); TGL-16C bench centrifuge (Shanghai' an pavilion scientific instruments factory); UV-2000 ultraviolet spectrophotometer (UNICO); FSH-2A adjustable high-speed homogenizer (Jintan medical instruments and instruments factory); XH-C vortex mixer (Tan City medical Instrument works).

1.2 methods

1.2.1 Molding method

A mouse uric acid nephropathy model is established by combining intragastric perfusion of yeast extract and intraperitoneal injection of oteracil potassium. Except for normal groups, the mice of other groups are intragastrically injected with 30g/kg of yeast extract every day for 15 days continuously, and are finally injected with 300mg/kg of oteracil potassium intraperitoneally for 1 day.

1.2.2 animal grouping and administration

60 male Kunming mice are taken and are adaptively fed for 3 days, and then are randomly divided into 6 groups according to the body weight, and each group comprises 10 mice, namely a blank group, a model group, an allopurinol group (5mg/kg/d), a baicalin low dose group (25mg/kg/d), a baicalin medium dose group (50mg/kg/d) and a baicalin high dose group (100 mg/kg/d). Allopurinol, baicalin and Potassium Oxonate are suspended in 0.8% CMC-Na aqueous solution. The administration was performed by intragastric administration (allopurinol was administered from 8d to 15d), the same volume of CMC-Na aqueous solution was administered to the group without administration, the 30g/kg yeast extract aqueous solution was administered after intragastric administration, the same volume of aqueous solution was administered to the group without administration, 300mg/kg potassium oxonate was intraperitoneally injected after the 15d intragastric administration of yeast extract, and blood was taken 1h later. The volume of the stomach perfusion and the abdominal cavity injection is 10 ml/kg.

1.2.3 measurement index

1h after the last model building, blood is taken from the vein behind the mouse eyes, whole blood is centrifuged at 3500rpm/min, and the supernatant is taken as serum and used for measuring the levels of serum uric acid, BUN and Cr. After the mice were sacrificed by dislocation, the kidneys, livers, spleens, and thymus were weighed, and the organ coefficient was (mg) organ mass/final mouse body mass (g). XOD activity in serum and liver was determined using the XOD kit.

1.2.4 statistical methods

The data were analyzed and plotted using GraphPad Prism 6, all using

Showing that the analysis between groups was statistically different using GraphPad Prism 6 for the t-test with P < 0.05.

1.3 results

1.3.1 Effect of baicalin on serum uric acid, Urea Nitrogen and creatinine in uric acid nephropathy mice

As shown in Table 1, the uric acid, uric acid nitrogen and creatinine levels in the model group are significantly increased (P <0.001) compared with the blank group, indicating that the uric acid nephropathy model is successfully established. Compared with a model group, the positive allopurinol can obviously reduce the uric acid level (P is less than 0.001), but has no obvious influence on the urea nitrogen and creatinine levels, which shows that the allopurinol has obvious effect on reducing the uric acid level but has no obvious effect on improving the renal function. Compared with the model group, the low, medium and high dose groups of baicalin can obviously reduce the serum uric acid level (the P is less than 0.01 in the low and medium dose groups and is less than 0.001 in the high dose group), and show certain dose dependence, which indicates that the baicalin has the function of reducing uric acid in vivo; the low, medium and high dose groups of baicalin obviously reduce the serum urea nitrogen and creatinine levels (the low dose group, P is less than 0.05; the medium and high dose groups, P is less than 0.01), and the baicalin has certain improvement effect on the nephropathy caused by the hyperuricemia.

Table 1 mouse serum uric acid, urea nitrogen, creatinine levels (n ═ 10)

Note: in comparison to the blank set, the data is,^***P<0.001; in comparison to the set of models,^#P<0.05，^##P<0.01，^###P＜0.001

1.3.2 Effect of baicalin on xanthine oxidase in uric acid nephropathy mice

As shown in Table 2, the XOD activity in the serum of the model group was not significantly increased (P >0.05) compared to that of the blank group. Compared with a model group, the allopurinol group has obviously reduced serum XOD activity (P < 0.001); baicalin dose groups had no significant effect on serum XOD activity (P > 0.05). Compared with the blank group, the liver XOD activity of the model group is remarkably increased (P <0.001), which indicates that hyperuricemia nephropathy can cause the liver XOD activity to be increased. Compared with the model group, the liver XOD activity of the allopurinol group is obviously reduced (P is less than 0.001), which shows that the allopurinol also has obvious inhibition effect on the XOD activity in the liver; the low, medium and high dose groups of baicalin can obviously inhibit liver XOD activity (the low dose group, P < 0.01; the medium dose group, P < 0.001; and the high dose group, P < 0.01).

TABLE 2 mouse serum and liver xanthine oxidase levels (n ═ 10)

Note: in comparison to the blank set, the data is,^***P<0.001; in comparison to the set of models,^##P<0.01，^###P＜0.001

1.3.3 Effect of baicalin on organ coefficients of uric acid nephropathy mice

As shown in Table 3, the liver coefficient, kidney coefficient and spleen coefficient of the model group were significantly increased (P <0.001) compared to the blank group, and the thymus coefficient was not significantly changed. Compared with a model group, allopurinol can further remarkably increase the liver coefficient (P is less than 0.01), reduce the kidney coefficient (P is less than 0.05) and further increase the spleen coefficient (P is less than 0.01); the low dose of baicalin has no obvious influence on the liver coefficient (P is more than 0.05), and the medium dose (P is less than 0.05) and the high dose (P is less than 0.001) of baicalin can obviously reduce the liver coefficient; the low, medium and high dose groups of baicalin can obviously reduce the kidney coefficient (the low dose group, P is less than 0.001; the medium dose group, P is less than 0.01; the high dose group, P is less than 0.001); the low, medium and high dose groups of baicalin can obviously reduce the spleen index (the P is less than 0.05 in the low dose group, and the P is less than 0.001 in the medium and high dose groups).

Table 3 mouse organ coefficient levels (n ═ 10)

2. effect of baicalin on sodium urate crystal induced injury of rat renal tubular epithelial cells

2.1 materials of the experiment

2.1.1 drugs and reagents

Uric acid (Sigma, batch number: BCBW1849, purity ≥ 99%); DMEM high-glucose cell culture medium (Hyclone, lot # 1559231); DMEM/F12 medium (Gibco, lot number 8118010); fetal bovine serum (Gibco, south America, lot number 42A 0378K); dimethylsulfoxide (Sigma, USA, lot number 20161109); phosphate Buffered Saline (PBS) (Shanghai double helix Biotechnology Co., Ltd., batch No: M0401A); penicillin, streptomycin (Hyclone, USA, batch number: 1677648); trypsin (containing 0.02% EDTA) (Gibco, USA, batch No.: 2042337). Sodium hydroxide and hydrochloric acid were purchased from Chinese herbs. TNF-alpha, IL-1 beta Elisa kits were purchased from Wuhan Sanying Biotech Ltd. NO kit, lot NO: 20191116, respectively; LDH kit, batch No.: 20191116, respectively; purchased from Nanjing institute of biological research. Baicalin (purity > 90%), batch number: C031B150902, kawa synergestic ltd. Dexamethasone (purity > 98%), batch number: l1904041, available from Shanghai Aladdin Biotech Ltd. The experimental water is self-made double distilled water in a laboratory.

2.1.3 Experimental instruments

Electronic analytical balance (model: AL204, Mettler-Torledo Corp.); vortex mixer (type: XH-C, Jiangsu province, jin Tan City medical instruments factory); enzyme-linked immunosorbent assay (model: EnSpire, Perkin Elmer, USA); a carbon dioxide incubator (model: W200IR, Cin Union (Beijing) instruments Co., Ltd.); a vertical pressure steam sterilizer (model: YXQ-LS-30SII, Shanghai Boxun industries, Ltd.) for medical facilities; centrifuge (model: TGL-16C, Shanghai' an pavilion scientific instruments factory); an ultrasonic cleaner (model: KQ3200E, ultrasonic instruments Co., Ltd., Kunshan city); clean bench (model: SW-CJ-1FD, Sujing Antai air technology Co., Ltd.).

2.1.4 cell lines

Rat tubular epithelial cells (NRK-52e) available from Kjekay GeneChemicals, Inc. (available from the Shanghai cell Bank of the Chinese academy of sciences of CTCC).

2.2 methods

2.2.1 preparation of sodium urate crystals

Adding 0.8g of uric acid into 160ml of water, heating until the water boils slightly, adding 1M of sodium hydroxide to dissolve the uric acid, adjusting the pH to 7 with dilute hydrochloric acid to separate out the uric acid, cooling, suspending, centrifuging the suspension repeatedly to obtain sodium urate crystals, suspending the sodium urate crystals with physiological saline, and sterilizing the suspension with high-pressure steam for later use.

2.2.2 cell treatment

Sucking old culture medium from a culture bottle full of cells, washing with PBS for 2 times, removing PBS, adding 1.5ml pancreatin, placing in an incubator for digestion for 2 minutes, adding the culture medium to stop digestion, and centrifuging to obtain the cells for later use.

2.2.3 Molding and administration

When the cells grew to 85%, the old medium was aspirated, rinsed once with PBS, and different drugs and sodium urate were added. The groups are as follows: blank group (complete medium), model group (complete medium of MSU at a final concentration of 2 mg/ml), dexamethasone group (complete medium of MSU at a final concentration of dexamethasone 40. mu. mol/L +2 mg/ml), baicalin low dose (complete medium of MSU at a final concentration of baicalin 20. mu. mol/L +2 mg/ml), baicalin medium dose (complete medium of MSU at a final concentration of baicalin 40. mu. mol/L +2 mg/ml), baicalin high dose (complete medium of MSU at a final concentration of baicalin 80. mu. mol/L +2 mg/ml), each experiment was repeated three times.

2.2.4 Biochemical index determination

And (3) taking cell supernatant, centrifuging at 1500rpm/min for 10 minutes, taking supernatant, subpackaging, and storing to-20 ℃ for measuring the contents of TNF-alpha, IL-1 beta, lactate dehydrogenase and NO.

2.3 results

2.3.1 Effect of baicalin on NRK-52e cell injury induced by sodium urate Crystal

(1) LDH release from cell supernatant

As shown in fig. 1, LDH release was significantly increased in the model group cells under MSU stimulation (P <0.01) compared to the blank group. Compared with the model group, the LDH release amount in the supernatant of the dexamethasone group is obviously reduced (P is less than 0.05), which indicates that the dexamethasone has a certain effect of resisting the injury of NRK-52e cells induced by MSU; the low and medium dose groups of baicalin have a reduced LDH release amount, but no significant difference (P is more than 0.05), which indicates that the concentration of the baicalin at 20 mu mol/L and 40 mu mol/L has no obvious effect on the up-regulation of LDH induced by MSU; the baicalin high-dose group can remarkably (P <0.01) reduce the release of LDH, and the summary shows that the baicalin has a certain effect of resisting the large-scale release of LDH caused by MSU and presents a certain dose-effect relationship.

(2) Cellular supernatant NO release

As shown in fig. 2, model group cell supernatants were significantly upregulated in NO release (P <0.001) with intervention of MSU compared to the blank group. NO significant reduction of NO in the cell supernatants of the dexamethasone group compared to the model group (P > 0.05); NO significant reduction of NO in cell supernatants of baicalin low dose groups (P > 0.05); the NO in the supernatant of baicalin and high-dose group is remarkably reduced (P < 0.05).

(3) TNF-alpha expression in cell supernatants

As shown in fig. 3, the expression level of TNF-a was significantly increased in the model group cell supernatants under MSU intervention (P <0.001) compared to the blank group. TNF-alpha in the cell supernatant of the dexamethasone group was significantly reduced compared to the model group (P < 0.01); the expression of TNF-alpha in the cell supernatant of the low, medium and high dose groups of baicalin is obviously reduced (P <0.05 or P < 0.01).

(4) Expression level of IL-1. beta. in cell supernatant

As shown in fig. 4, the expression level of IL-1 β in the supernatant of model group cells was significantly increased compared to the blank group (P < 0.01). Compared with the model, the expression level of the IL-1 beta of the supernatant fluid of the dexamethasone cell is obviously reduced (P < 0.01); the expression level of IL-1 beta in the supernatant of the low-dose baicalin group is not significantly reduced (P is more than 0.05); the IL-1 beta expression of the supernatant of the medium and high dose group cells of the baicalin is remarkably reduced (P is less than 0.05).

Conclusion

Allopurinol is a first-line uric acid generation inhibiting drug widely applied clinically, is an isomer of hypoxanthine, is a suicide substrate of XOD, is a xanthine oxidase competitive inhibitor, and can inhibit oxidation of the hypoxanthine and the xanthine by the XOD to generate uric acid, so that the uric acid level is reduced. Allopurinol is widely used for treating hyperuricemia or related diseases clinically, and although the effect of lowering uric acid of allopurinol is remarkable, a large number of adverse reactions are reported clinically, such as: including impairment of liver function, kidney function, skin rash and hypersensitivity syndrome, wherein the hypersensitivity syndrome can cause a certain lethality, and the above adverse reactions significantly limit the clinical application of allopurinol. Baicalin is a flavonoid compound mainly existing in traditional Chinese medicine scutellaria baicalensis, and has wide pharmacological action and small toxic and side effects. In the experiment, compared with a model group, the baicalin can obviously reduce the serum uric acid, uric acid nitrogen and creatinine levels of a uric acid nephropathy mouse, and presents a certain dose dependence, which indicates that the baicalin can not only reduce the uric acid level, but also improve the renal function reduction of the mouse caused by hyperuricemia. Compared with a model group, the baicalin can obviously inhibit the XOD activity of the liver, which indicates that the baicalin reduces the serum uric acid level by inhibiting the XOD activity. Compared with a model group, the baicalin can reduce the increase of the coefficients of the kidney, the liver and the spleen of a mouse caused by hyperuricemia, and the result shows that the baicalin can effectively reduce the damage of the kidney, the liver and the spleen caused by the hyperuricemia, and the baicalin can protect the kidney damage caused by the hyperuricemia and also has a protective effect on the liver and the spleen.

In patients with long-term hyperuricemia, the deposition of urate crystals can be caused to the renal tubules, so that the acute inflammatory injury of the kidney is caused, and the symptoms are shown as follows: acute pain in the kidney area, abdominal fever, proteinuria, and the like. For acute attack of hyperuricemia nephropathy with urate deposition, anti-inflammatory treatment is mainly adopted clinically, and the commonly used medicines comprise dexamethasone, colchicine and the like. The common dose of colchicine and the dose of toxic agents are relatively close, so the incidence rate of adverse reactions is high, and the adverse reactions mainly include diarrhea, vomiting, nausea and hepatocyte necrosis, so the colchicine is rarely used clinically at present; dexamethasone is not suitable for long-term use and is easy to generate drug dependence and drug-induced diseases. The invention takes NRK-52e cells as a research object, adopts sodium urate crystals to prepare a model of sodium urate crystals damaging renal tubule cells, adopts baicalin and a positive drug dexamethasone for intervention, and adopts indexes related to NO, LDH, TNF-alpha and IL-1 beta inflammation to observe the effect of the drug. Compared with the blank group, NO, LDH, TNF-alpha and IL-1 beta in the cell supernatant of the model group are obviously increased, which indicates that the sodium urate crystal initiates the generation of inflammatory injury of renal tubular epithelial cells, and indicates that the model is successfully established. Compared with a model group, the baicalin can obviously reduce the levels of NO, LDH, TNF-alpha and IL-1 beta to different degrees, and the result shows that the baicalin can protect the inflammatory injury of renal tubular epithelial cells caused by the stimulation of sodium urate.

In conclusion, baicalin can play roles in reducing uric acid and protecting the kidney function of mice with uric acid nephropathy by inhibiting xanthine oxidase and resisting inflammation.

3. Protection effect of baicalin on hyperuricemic kidney injury mice

3.1 materials

3.1.1 animals

SPF-level Kunming mice, male, with a weight of 20-22 g, provided by the Experimental animal center of Huazhong university of science and technology. Animal production license number: SCXK (jaw) 2016-.

3.1.2 drugs and reagents

Allopurinol (batch No. Y27O8C46913), available from shanghai source, lobe biotechnology ltd; baicalin (purity not less than 90%) purchased from Sichuan cooperative pharmaceutical corporation; potassium oxonate (batch No. P137112), available from Shanghai Allantin Biotech, Inc.; yeast extract (lot 20170802), available from biotechnology responsibility of Obo Star, Beijing; uric Acid (UA) assay kit (lot 20181017), xanthine oxidase test kit (lot 20180920), urea nitrogen (Blood urea nitrogen, BUN) kit (lot 20181024), Creatinine (Cr) kit (lot 20181022), Superoxide dismutase (SOD) kit, Malondialdehyde (MDA) kit, Glutathione peroxidase (GPx) kit, Catalase (Catalase, CAT) kit, coomassie brilliant blue kit (lot 20180921), all available from tokyo institute of biotechnology; dimethyl sulfoxide (lot 20161109) available from national pharmaceutical group chemical Co.

3.1.3 instruments

AL204 electronic analytical balance (mettler-toledo corporation); SPS2001F electronic balance (aohaus corporation); TGL-16C bench centrifuge (Shanghai' an pavilion scientific instruments factory); UV-2000 ultraviolet spectrophotometer (ewnikov); FSH-2A Adjustable high speed homogenizer (Instrument works, gold Tan).

3.2 methods

3.2.1 animal grouping and administration

50 male Kunming mice were divided into 5 groups of 10 mice each by body weight after adaptive feeding for 3 d. The group was divided into a normal group, a model group, an allopurinol group (5mg/kg), a baicalin gavage group (i.g., 50mg/kg), and a baicalin intraperitoneal injection group (i.p., 50 mg/kg). Beginning to administer the medicine regularly in the morning at 1d, mice in a normal group and a model group are respectively administered with normal saline through intragastric administration, baicalin i.g group and baicalin i.p group are respectively administered with corresponding dose of baicalin, continuous 15d, 0.5 percent CMC-Na is intragastric administered at 8d before an allopurinol group, and finally allopurinol is administered through intragastric administration at 7 d. The volume of the medicine for intraperitoneal injection is 0.15ml/10g, and the volume of the medicine for intragastric administration is 0.2ml/10 g.

3.2.2 establishment of hyperuricemia Kidney Damage animal model

A mouse hyperuricemia nephropathy model is established by adopting yeast cream intragastric administration in combination with oteracil potassium intraperitoneal injection. Except for the normal group, the other groups of mice were subjected to intragastric injection of yeast cream (30g/kg) every day for 15 days continuously and finally 1 day of intraperitoneal injection of potassium oxonate (300mg/kg), except for the allopurinol group, the intragastric injection volume of the other groups was 0.2ml/10g, the intraperitoneal injection volume was 0.15ml/10g and the allopurinol intragastric injection volume was 0.1ml/10 g. 1h after the drug treatment, except the normal group, the other groups are perfused with the yeast extract solution, and finally the potassium oxonate solution is injected into the abdominal cavity after the 1d of the yeast extract is perfused.

3.2.3 preparation of pharmaceutical solutions

Dissolving baicalin in DMSO, diluting with physiological saline to obtain baicalin suspension of 5mg/mL (DMSO content is less than 5%).

0.5 percent of CMC-Na is used as a solvent to prepare 0.5mg/mL allopurinol suspension and 3g/mL yeast extract suspension, and 20mg/mL oteracil potassium suspension respectively.

3.2.4 Biochemical index determination

And (3) taking blood from eyeballs of mice of each group 1 hour after the drug administration and the model building, centrifuging at 3500rpm for 10min, taking supernatant, storing at-20 ℃, measuring the levels of UA, BUN and Cr in serum, killing the mice by dislocation of cervical vertebrae, weighing the weight of whole kidney, liver, thymus and spleen, and calculating the organ index. Liver was taken, homogenized mechanically according to kit instructions and liver XO levels were determined. And (3) mechanically homogenizing the kidney of the same side of the mouse according to the instruction of the kit, and measuring the oxidative stress level of the kidney of the mouse.

3.3 results

3.3.1 Effect of baicalin on Kidney function of hyperuricemia mice

The results are shown in Table 4. As can be seen from Table 4, compared with the normal group, the levels of serum uric acid, urea nitrogen and creatinine of the mice in the model group are remarkably increased (P is less than 0.01), which indicates that the hyperuricemia of the mice can be caused by combining yeast extract with oteracil potassium and the kidney is damaged; compared with a model group, the serum uric acid level of mice in the allopurinol group is extremely obviously reduced (P <0.01), which indicates that the allopurinol can reduce the serum uric acid of the mice, but the serum urea nitrogen and creatinine level of the mice have no statistical significance (P >0.05), and indicates that the allopurinol has no protective effect on the kidney; the baicalin i.g group significantly reduced serum UA levels (P <0.05), but had no significant effect on BUN and Cr (P > 0.05). The baicalin i.p group remarkably reduces serum UA level (P <0.05), extremely remarkably reduces BUN level (P <0.01) and remarkably reduces Cr level (P <0.05), which indicates that the intraperitoneal injection of baicalin has better kidney protection effect while reducing serum UA level.

Table 4 effect of baicalin on serum uric acid, urea nitrogen, creatinine levels in uric acid nephropathy mice (x ± s, n ═ 10)

Note: andthe comparison of the normal group is carried out,^*P<0.05，^**P<0.01; in comparison with the set of models,^#P<0.05，^##P<0.01. i.g, intragastric administration; i.p, i.p. intraperitoneal injection.

3.3.2 Effect of baicalin on visceral index of uric acid nephropathy mice

The results are shown in Table 5. As can be seen from Table 5, the renal index of the model group is significantly increased (P <0.01) compared with that of the normal group, which indicates that the kidney of the mice with hyperuricemia is increased, and the modeling mode has certain damage to the kidney. Compared with the model group, the kidney index of mice in the allopurinol group is obviously reduced (P < 0.01); the kidney index of mice in the i.g group of baicalin is obviously reduced (P is less than 0.01), and the kidney index of mice in the i.p group of baicalin is obviously reduced (P is less than 0.05), so that the baicalin has a certain protection effect on the kidney damage of the mice with hyperuricemia nephropathy. Compared with the normal group, the kidney index of mice in the baicalin i.g group and the baicalin i.p group has no statistical difference (P is more than 0.05), which indicates that the kidney index of the mice induced by high uric acid can be increased and recovered to the level of normal mice by virtue of the intragastric administration and intraperitoneal injection of the baicalin.

Table 5 effect of baicalin on mouse organ index (mg/g) (x ± s, n ═ 10)

In comparison with the normal group,^*P<0.05，^**P<0.01; in comparison with the set of models,^#P<0.05，^##P<0.01。

3.3.3 Effect of baicalin on the Activity of liver XO in uric acid nephropathy mice

The results are shown in Table 6. As can be seen from table 6, the XO activity was significantly increased in the model group (P <0.01) compared to the normal group, indicating that hyperuricemia mice were able to increase the XO activity. Compared with the model group, the XO activity of the allopurinol group is greatly reduced (P <0.01), and the XO activity of the baicalin i.g group and the baicalin i.p group is both significantly reduced (P < 0.05).

TABLE 6 Effect of baicalin on mouse liver xanthine oxidase Activity (x. + -.s, n ═ 10)

Note: in comparison with the normal group,^*P<0.05，^**P<0.01; in comparison with the set of models,^#P<0.05，^##P<0.01。

3.3.4 Effect of baicalin on Kidney oxidative stress in mice with hyperuricemic nephropathy

The results are shown in Table 7. As can be seen from table 7, compared with the normal group, the renal SOD level of the model group mice is significantly reduced (P <0.05), and MDA is significantly increased (P <0.01), indicating that the higher serum uric acid level breaks the oxidative stress balance of the mouse kidney, increasing the oxidative stress level, which further results in severe kidney loss. Compared with the model group, the allopurinol group, baicalin i.g group and baicalin i.p group all improved the decrease in SOD level and the increase in MDA level caused by hyperuricemia to different degrees (P <0.05, P < 0.01). The results show that baicalin has certain curative effect on the increase of the oxidative stress level caused by high uric acid, and can protect the kidney injury caused by the increase of the oxidative stress level.

Table 7 effect of baicalin on the level of oxidative stress in kidney of hyperuricemic nephropathy mouse (x ± s, n ═ 10)

conclusion

In recent years, the document reports that baicalin can inhibit the activity of XO in vitro, but the in vivo research report on hyperuricemia nephropathy is not found. The research result shows that the levels of serum uric acid of a mouse with hyperuricemia nephropathy can be obviously reduced by virtue of the administration of baicalin through gastric gavage and abdominal cavity, and the levels of serum urea nitrogen and creatinine can be obviously reduced by virtue of the injection of baicalin through abdominal cavity, so that the damage of the kidney function of the mouse caused by hyperuricemia can be reversed by virtue of the baicalin, and the kidney function of the mouse with hyperuricemia is further protected. And baicalin can be used for reducing the rise of mouse kidney index caused by hyperuricemia through gastric lavage and intraperitoneal injection, and has a protective effect on mouse kidney. Imbalance of oxidative stress is an important way for kidney injury caused by hyperuricemia, and the change of the kidney oxidative stress index of mice caused by hyperuricemia can be improved by virtue of both the intragastric administration of baicalin and the intraperitoneal injection. Hyperuricemia can cause kidney injury through multiple ways, such as induced inflammation, oxidative stress reaction, endothelial dysfunction, interstitial injury, aggravated renal fibrosis and the like, and baicalin has pharmacological effects of anti-inflammation, antioxidation and the like.

Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

Claims

1. Application of baicalin in preparing medicine for preventing and/or treating asymptomatic hyperuricemia and/or uric acid nephropathy.

2. The use of claim 1, wherein the medicament further comprises allopurinol.

3. The use of claim 1, wherein the uric acid nephropathy comprises acute uric acid nephropathy, chronic uric acid nephropathy, and uric acid nephrolithiasis.

4. The use of claim 1, wherein the medicament is in a dosage form of oral or injectable.

5. The use according to claim 1, wherein the medicament is administered in a dose of 20-200mg/kg/d, based on the weight of baicalin.

6. Application of baicalin in preparing medicine for reducing serum uric acid, urea nitrogen and creatinine level is provided.

7. Application of baicalin in preparing TNF-alpha, IL-1 beta, NO and lactate dehydrogenase inhibitors is provided.