Uses of Ten-Flavor Frankincense Powder in the Prevention and Treatment of Kidney Diseases

Abstract

This invention relates to the field of traditional Chinese medicine, specifically to the use of a ten-ingredient frankincense powder in the prevention and treatment of kidney diseases. The ten-ingredient frankincense powder exhibits antioxidant effects by reducing ROS and MDA levels and increasing SOD activity. It also inhibits the expression of inflammatory factors IL-1β and TNF-α, as well as pro-apoptotic genes Bax, Caspase-3, and Caspase-9, thus exerting anti-inflammatory and anti-apoptotic effects. It effectively prevents and treats kidney damage, and its mechanism may be related to the upregulation of the Pink1 / Parkin signaling pathway and activation of mitophagy.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, and in particular to the use of Tibetan medicine Ten-Flavor Frankincense Powder in the prevention and treatment of kidney diseases. Background Technology

[0002] Clinical manifestations of kidney disease include the clinical symptoms of the kidney disease itself and symptoms in various systems caused by impaired kidney function, including abnormal urine color, abnormal urine volume, abnormal urination, edema, and fatigue. Secondary kidney disease may also present with manifestations of the primary disease and damage to other organs, such as rashes, joint pain, oral ulcers, and hair loss.

[0003] Kidney diseases often present as certain clinical syndromes, but these syndromes may overlap. The same clinical syndrome can manifest as different pathological types of kidney disease, and the same pathological type of kidney disease can also manifest as different clinical syndromes. Common clinical syndromes of kidney disease include nephrotic syndrome, nephritis syndrome, asymptomatic hematuria and / or proteinuria (also known as latent glomerulonephritis), acute kidney injury, and chronic kidney disease.

[0004] Primary glomerulonephritis is a common glomerular disease, clinically manifesting as proteinuria, hematuria, edema, and hypertension. However, the severity varies from patient to patient. Many patients present with edema as the initial symptom; mild cases may only experience slight swelling of the eyelids and face upon waking, and slight edema of the lower extremities in the afternoon, which usually disappears after rest. Clinically, it is mainly divided into four categories: acute glomerulonephritis, rapidly progressive glomerulonephritis, chronic glomerulonephritis, and latent glomerulonephritis.

[0005] Secondary nephropathy is a kidney disease with a clear cause, including hypertensive nephropathy, diabetic nephropathy, epileptic nephritis, lupus nephritis, hepatic kidney damage, primary small vessel vasculitis kidney damage, rheumatoid arthritis kidney damage, acute rheumatic fever kidney damage, gouty kidney damage, systemic lupus erythematosus kidney damage, and malignant tumor kidney damage.

[0006] These diseases typically cause kidney damage, primarily affecting the glomeruli, and are often characterized by clinical manifestations and pathological changes primarily involving glomerular damage.

[0007] Interstitial nephritis, also known as tubulointerstitial nephritis, is a disease caused by various factors leading to chronic damage to the renal tubules and interstitial tissues. Clinically, it is often divided into acute interstitial nephritis and chronic interstitial nephritis. Acute interstitial nephritis is characterized by the rapid onset of inflammatory cell infiltration in the renal interstitium, interstitial edema, and varying degrees of tubular damage accompanied by renal insufficiency, all caused by various factors within a short period of time. Clinical manifestations can range from mild to severe. Most cases have a clear cause, and with removal of the cause and timely treatment, the disease can be cured or the condition can be reversed to varying degrees. The pathological manifestations of chronic interstitial nephritis are mainly characterized by renal interstitial fibrosis, interstitial mononuclear cell infiltration, and renal tubular atrophy.

[0008] Hereditary nephropathy refers to nephrotic syndromes caused by mutations in genes encoding proteins that make up the glomerular filtration barrier or other related genes. Clinically, the vast majority of cases present as hormone-resistant nephrotic syndrome. Symptoms include kidney damage, sensorineural hearing loss, and the formation of a cone-shaped lens in the eye. Early kidney failure and death are common, and women are prone to premature birth and miscarriage during pregnancy.

[0009] Nephrotic syndrome (NS) is a clinical syndrome comprised of a group of glomerular diseases with similar clinical manifestations but different etiologies and pathological changes. It commonly presents with typical symptoms such as massive proteinuria, severe edema, hyperlipidemia, and hypoproteinemia, and can lead to renal impairment, bacterial infection, and protein and metabolic disorders.

[0010] Nephritis syndrome is a clinical diagnostic type of kidney disease. It is mainly characterized by proteinuria, not exceeding 3.5g / 24h; decreased serum albumin, but not lower than 30g / L; and may also present with edema or hyperlipidemia, as well as hypertension.

[0011] Acute kidney injury (AKI) is a clinical syndrome caused by a rapid decline in kidney function over a short period of time due to various etiologies. It is characterized by a decrease in glomerular filtration rate, accompanied by retention of nitrogenous products such as creatinine and urea nitrogen, and disturbances in water, electrolyte and acid-base balance. In severe cases, multiple system syndrome may occur.

[0012] The decline in kidney function caused by chronic kidney disease (CKD) is irreversible and gradually progresses to end-stage renal disease. Currently, more than 500 million people worldwide suffer from CKD to varying degrees.

[0013] Oxidative stress, inflammation, and mitochondrial dysfunction are considered important mechanisms in many kidney diseases. In chronic kidney disease (CKD), identifying damaged mitochondria as targets to regulate the activation and inhibition of mitophagy, and further modulating the body's oxidative stress and inflammatory responses, could serve as an effective approach to treating kidney tissue damage. Autophagy is a mechanism for maintaining the dynamic balance of energy and resources in the body. Mitophagy, through the degradation and removal of damaged and dysfunctional mitochondria by intracellular lysosomes, facilitates the renewal of new mitochondrial organelles, maintains cellular homeostasis, and inhibits cell damage. When the body experiences oxidative stress, ROS produced by mitochondria cause DNA damage, enzyme oxidation and inactivation, leading to mitochondrial dysfunction. Recent studies have found that autophagy is associated with the pathogenesis of kidney diseases such as CKD, acute kidney injury, drug-induced kidney damage, hereditary kidney diseases, and diabetic nephropathy, as well as kidney aging.

[0014] Traditional Chinese medicine (TCM) has advantages in treating chronic kidney disease due to its diverse active ingredients, including good efficacy, high safety, and few adverse reactions. Numerous studies have shown that TCM can mediate the PINK / Parkin signaling pathway to regulate mitophagy, controlling renal oxidative stress, inflammation, cell death, tissue damage, and the progression of kidney disease. The Tibetan medicine Ten-Flavor Frankincense Powder is a classic Tibetan prescription composed of ten Tibetan herbs, including frankincense, costus root, broad-leaved vine, cassia seed, and chebula. The *Four Medical Classics* record its effects of dispelling wind and dampness, and drying yellow fluid. Traditionally, it is used for rheumatoid arthritis, joint redness and swelling caused by gout, and eczema caused by excessive yellow fluid. Modern research indicates that the Ten-Flavor Frankincense Powder has sedative, anti-inflammatory, and uric acid-lowering effects. It is also effective in treating gouty arthritis, rheumatoid arthritis, and other rheumatic diseases. Summary of the Invention

[0015] The purpose of this invention is to provide a new use of the Ten-Flavor Frankincense Powder in the prevention and treatment of kidney diseases, and to provide a pharmacodynamic basis for the development of new adaptations of the Ten-Flavor Frankincense Powder.

[0016] The kidney diseases of this invention are kidney tissue damage diseases, including primary glomerulonephritis, secondary nephropathy, interstitial nephritis, hereditary nephropathy, nephrotic syndrome, nephritis syndrome, acute kidney injury, and chronic kidney disease.

[0017] Among them, primary glomerulonephritis includes acute glomerulonephritis, rapidly progressive glomerulonephritis, chronic glomerulonephritis, and latent glomerulonephritis; secondary nephropathy includes lupus nephritis, diabetic nephropathy, vasculitis-related nephropathy, and hyperuricemic nephropathy; interstitial nephritis includes acute interstitial nephritis and chronic interstitial nephritis; and acute kidney injury refers to renal ischemia and renal tubular epithelial cell damage caused by nephrotoxic substances.

[0018] The prescription composition of the Ten-Flavor Frankincense Powder of the present invention is as follows: 100 parts frankincense, 150 parts chebula, 80 parts cassia seed, 100 parts chebula root, 80 parts okra seed, 120 parts amla seed, 85 parts costus root, 100 parts broad-leaved vine, 80 parts baxiaga, and 50 parts zaoxun paste. Furthermore, the Ten-Flavor Frankincense Powder of the present invention is a pharmaceutical preparation prepared from the above prescription composition and a pharmaceutical carrier.

[0019] Animal experiments have shown that Shiwei Frankincense Powder can reduce ROS and MDA content, increase SOD activity to exert antioxidant effects, inhibit the expression of inflammatory factors IL-1β and TNF-α and pro-apoptotic genes Bax, Caspase-3 and Caspase-9 to exert anti-inflammatory and anti-apoptotic effects, and effectively prevent and improve kidney damage. Its mechanism may be related to the upregulation of the Pink1 / Parkin signaling pathway to activate mitophagy. Attached Figure Description

[0020] Figure 1These are pathological images of rat kidney tissue under an optical microscope (HE, ×400) from each group in Example 2;

[0021] Figure 2 These are images of cell apoptosis in rat kidney tissue from each group in Example 2 under an optical microscope (TUNLE, ×400).

[0022] Figure 3 This refers to the apoptosis rate of rat kidney tissue cells in each group in Example 2;

[0023] Figure 4 The expression of Caspase-3, Caspase-9, BAX, and Bcl-2 proteins and mRNA in rats in each group of Example 2;

[0024] Figure 5 This refers to the expression of IL-1β and TNF-α proteins and mRNA in rats in each group in Example 2;

[0025] Figure 6 This refers to the expression of PINK1, Parkin, and LC-3Ⅱ proteins in rats in each group of Example 2.

[0026] In the figure, A: blank group; B: model group; C: low-dose group of Shiwei Ruxiang San (a traditional Chinese medicine formula); D: medium-dose group of Shiwei Ruxiang San; E: high-dose group of Shiwei Ruxiang San; F: allopurinol group; VS blank group, **p<0.05; VS model group, ## p < 0.05; VS. low-dose group of Shiwei Ruxiang San, ▲▲ p < 0.05. Detailed Implementation

[0027] The present invention will now be further described with reference to specific embodiments. The following embodiments are intended to illustrate the present invention and not to further limit the present invention.

[0028] Effects of Example 1 on a rat model of chronic glomerulonephritis

[0029] Sixty SD rats, weighing 150±10g, were acclimatized for 3 days. Ten rats were randomly selected as the control group, and the rest as the model group. The model group rats were injected intravenously with doxorubicin (7mg / kg) once via tail vein to establish a chronic glomerulonephritis model according to literature methods. 24-hour urinary protein was measured weekly; a urinary protein content >100mg was considered a successful model. The model group rats were randomly divided into a model group and low, medium, and high dose groups of Ten-Flavor Frankincense Powder (TFRXP) (the dosages were calculated as 200, 400, and 800mg / kg based on the animal-to-human surface area ratio), with 10 rats in each group. All groups received the corresponding drugs via gavage. The control and model groups received the same volume of pure water once daily. The administration volume was 10mL / kg. The rats were weighed every 4 days, and the gavage volume was adjusted accordingly, for 14 consecutive days.

[0030] One hour after the last administration, rats were placed in metabolic cages, and 24-hour urine was collected. Urinary protein levels were measured according to the kit instructions. Twenty-four hours after the last administration, rats were anesthetized with 3% isoflurane, and blood was collected from the abdominal aorta. The blood was centrifuged at 3500 rpm for 10 minutes at 4°C, and serum was collected. Serum urea and creatinine levels were measured by ELISA. 200 mg of the left kidney was collected, and after removing blood and surface moisture, a 10% tissue homogenate was prepared. The supernatant was collected by centrifugation, and the levels of inflammatory factors TNF-α and IL-1β in the kidney tissue were measured by ELISA.

[0031] Experimental results:

[0032] 1. Effects of TFRXP on renal function in rats

[0033] Compared with the control group, the serum urea and SCr levels and 24-hour urinary protein levels in the model group rats were significantly increased (p < 0.05). Compared with the model group, the serum urea, creatinine, and 24-hour urinary protein levels in the low, medium, and high dose TFRXP groups were significantly decreased (p < 0.05), as shown in Table 1.

[0034] Table 1. Effects of 24-hour urinary protein and serum Urea and SCr levels in rats with chronic glomerulonephritis. n = 10

[0035]

[0036] VS blank group, *p<0.05; VS model group, # p < 0.05.

[0037] 2. Effects of TFRXP on inflammatory factors in rat kidney tissue

[0038] Compared with the blank group, the levels of IL-1β and TNF-α in the kidney tissue of rats in the model group were significantly increased (p<0.05), indicating that the kidney tissue of rats showed obvious inflammation after successful modeling. Compared with the model group, the levels of IL-1β and TNF-α in the kidney tissue of rats in the low, medium and high dose TFRXP groups were significantly decreased (p<0.05), indicating that TFRXP has anti-inflammatory effects (see Table 2).

[0039] Table 2. Effects on the levels of inflammatory factors in the renal tissue of rats with chronic glomerulonephritis. n = 10

[0040]

[0041] VS blank group, *p<0.05; VS model group, # p < 0.05.

[0042] In summary, Shiwei Ruxiang San can inhibit the levels of inflammatory factors in rats with glomerular inflammation, reduce the content of urinary protein, reduce the levels of blood urea and serum creatinine, improve renal function, and alleviate renal inflammatory response.

[0043] Example 2 Effects on rats with hyperuricemic kidney damage

[0044] Forty-eight rats were randomly divided into six groups: a control group, a model group, low-, medium-, and high-dose TFRXP groups (200, 400, and 800 mg / kg, respectively), and an allopurinol group (10 mg / kg), with eight rats in each group. During the experiment, except for the control group, all other groups were administered a suspension of potassium oxonate (750 mg / kg) and uric acid (300 mg / kg) (0.5% CMC-Na) by gavage daily. The control group was given the same volume of 0.5% CMC-Na solution once a day for 28 consecutive days to establish a rat model of gouty nephropathy. Simultaneously with model establishment, all groups were administered the drugs by gavage, while the control and model groups were given the same volume of pure water once a day. The drug administration volume was 10 mL / kg, and the rats were weighed every 4 days to adjust the gavage volume accordingly.

[0045] After 28 days, the subjects were kept on a fasting schedule but allowed free access to water for 24 hours. Blood was drawn from the abdominal vein under intraperitoneal anesthesia with 3% sodium pentobarbital. After the blood was allowed to stand for 2 hours, it was centrifuged at 3500 rpm for 10 minutes at 4°C to obtain serum samples. The serum was then frozen and stored at -80°C for later use. Liver and kidney tissues were rapidly separated on an ice table. The liver and left kidney tissues were stored at -80°C for later testing, while the right kidney was placed in a fixative.

[0046] Serum uric acid (UA), serum creatinine (SCr), and serum blood urea nitrogen (BUN) were detected by ELISA in rats. Histopathological changes in rat kidney tissue were observed by HE staining. Renal cell apoptosis was observed by TUNEL staining. Rat kidney tissue was collected, and the levels of superoxide dismutase (SOD), malondialdehyde (MDA), and reactive oxygen species (ROS) were detected according to the kit instructions. The expression of renal inflammatory cytokines, apoptosis-related genes, and mitophagy-related genes was measured by Western blot; the mRNA levels of renal inflammatory cytokines and apoptosis-related genes were measured by real-time quantitative PCR.

[0047] Experimental results:

[0048] 1. Effects of TFRXP on renal function in rats

[0049] Compared with the control group, the serum UA, BUN, and SCr levels in the model group rats were significantly increased (p < 0.05), indicating that the GN rat model was successfully established. Compared with the model group, the serum uric acid, urea nitrogen, and creatinine levels in the low, medium, and high dose TFRXP groups and the allopurinol group rats were significantly decreased (p < 0.05), as shown in Table 3.

[0050] Table 3 Effects on serum uric acid, creatinine and urea nitrogen levels in rats n=8

[0051]

[0052]

[0053] VS blank group, *p<0.05; VS model group, # p < 0.05; VS. low-dose group of Shiwei Ruxiang San, ▲ p < 0.05.

[0054] 2. Pathological results

[0055] HE-stained histopathological images showed that in the control group, the glomeruli and tubules of rats were morphologically normal, with clear structures, orderly cell arrangement, and no inflammatory infiltration. In the model group, the glomeruli showed no obvious pathological changes, but a large number of tubules were significantly enlarged, with abundant urate crystals in the renal interstitium and visible inflammatory cell infiltration. Compared with the model group, the medium- and high-dose TFRXP groups and the allopurinol group showed enlargement of a few tubules, reduced distribution of inflammatory cells, and reduced urate crystals, while the low-dose TFRXP group showed no significant improvement. Figure 1 .

[0056] 3. Study on the effect and mechanism of TFRXP on apoptosis of rat kidney tissue cells

[0057] TUNEL staining showed a small amount of apoptosis in the blank group, while a large number of apoptotic cells were observed in the kidney tissue cells of the model group. Compared with the model group, apoptosis was significantly reduced in the low, medium, and high dose TFRXP groups and the allopurinol group, and the reduction in apoptosis was more pronounced in the medium and high dose TFRXP groups than in the low dose group. Figure 2 .

[0058] Compared with the control group, the apoptosis rate of renal tissue cells in the model group was significantly increased (p < 0.05); compared with the model group, the apoptosis rate of renal tissue cells in the low-, medium-, and high-dose TFRXP groups and the allopurinol group was decreased (p < 0.05). Figure 3 .

[0059] Compared with the control group, the expression of Bcl-2 in the model group rats was significantly decreased (p < 0.05), while the expression of Bax, Caspase-9, and Caspase-3 was significantly increased (p < 0.05). Compared with the model group, the expression of Bcl-2 in the low-, medium-, and high-dose TFRXP groups and the allopurinol group was significantly increased (p < 0.05), while the expression of Bax, Caspase-9, and Caspase-3 was significantly decreased (p < 0.05). Furthermore, the expression of Bcl-2, Bax, Caspase-9, and Caspase-3 in the renal tissue of the medium- and high-dose TFRXP groups was significantly different from that in the low-dose group (p < 0.05). These results indicate that renal cell apoptosis is an important characteristic of GN progression, and TFRXP significantly inhibits renal cell apoptosis. Figure 4 .

[0060] 4. Effects of TFRXP on oxidative stress and inflammatory response levels in rats

[0061] Compared with the control group, the SOD level in the kidneys of rats in the model group was significantly decreased (p<0.05), while the MDA and ROS levels were significantly increased (p<0.05), indicating that the kidney tissue of GN rats showed a significant oxidative stress response. Compared with the model group, the SOD level in the kidneys of rats in the low-, medium-, and high-dose TFRXP groups and the allopurinol group was significantly increased (p<0.05), while the MDA and ROS levels were significantly decreased (p<0.05). Compared with the low-dose TFRXP group, the SOD level in the kidney tissue of the medium- and high-dose groups was significantly increased (p<0.05), while the MDA and ROS levels were significantly decreased (p<0.05), indicating that TFRXP may exert its effect through antioxidation. See Table 4.

[0062] Compared with the control group, the expression of IL-1β and TNF-α in the kidney tissue of rats in the model group was significantly increased (p < 0.05), indicating that the kidney tissue of rats showed obvious inflammation after successful modeling. Compared with the model group, the expression of IL-1β and TNF-α in the kidney tissue of rats in the low, medium, and high dose TFRXP groups and the allopurinol group was decreased, and the differences were significant (p < 0.05). The expression of IL-1β and TNF-α in the kidney tissue of the medium and high dose TFRXP groups was significantly lower than that of the low dose group (p < 0.05), indicating that TFRXP may have anti-inflammatory effects. Figure 5 .

[0063] Table 4. SOD activity, MDA and ROS levels in rat kidney tissue of each group n=8

[0064]

[0065] VS blank group, *p<0.05; VS model group, # p < 0.05; VS. low-dose group of Shiwei Ruxiang San, ▲p < 0.055; Effect of TFRXP on the mitochondrial autophagy pathway in rat kidneys

[0066] Compared with the control group, the expression levels of PINK1, Parkin, and LC-3II, all related to mitochondrial autophagy, were significantly decreased in the model group mice (p < 0.05), suggesting that mitochondrial autophagy in rat kidneys is inhibited under uric acid stimulation. Compared with the model group, the expression levels of PINK1, Parkin, and LC3II, all related to mitochondrial autophagy, were increased in the low, medium, and high dose TFRXP groups (p < 0.05), suggesting that TFRXP can activate mitophagy to some extent. The promoting effect of the medium and high dose TFRXP groups was more significant than that of the low dose group (p < 0.05), while there was no difference between the medium and high dose groups. Figure 6 .

[0067] In summary, the Ten-Flavor Frankincense Powder has antioxidant effects by reducing ROS and MDA content and increasing SOD activity, and it also has anti-inflammatory and anti-apoptotic effects by inhibiting the expression of inflammatory factors IL-1β and TNF-α and pro-apoptotic genes Bax, Caspase-3, and Caspase-9, thus effectively preventing and improving kidney damage.

[0068] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The use of Ten-Flavor Frankincense Powder in the preparation of drugs for the prevention and treatment of kidney diseases, characterized in that, The kidney disease is chronic glomerulonephritis. The prescription composition of the Ten-Flavor Frankincense Powder is as follows: 100 parts frankincense, 150 parts chebula, 80 parts cassia seed, 100 parts chebula fruit, 80 parts okra seed, 120 parts amla fruit, 85 parts costus root, 100 parts broad-leaved vine, 80 parts baxiaga, and 50 parts zaoxun paste.

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