Study On The Anti-kidney Stone Effect Of Hovenia Dulcis In Vivo And In Vitro

Kidney stones are a common multiple disease of the urinary system worldwide,and their incidence has gradually increased in recent years.The treatment of kidney stones is generally surgical treatment,but this will cause irreversible damage to the body;natural medicinal plants not only the curative effect is good,but also the side effects are small,so natural medicinal plants have certain development potential in anti-calculus activity.Hovenia dulcis,the official name is Hovenia acerba Lindl,which is the fruit of Hovenia dulcis in the rhamnaceae family.As one of the natural medicine and food homologous plants,it has good pharmacological activities such as anti-alcoholism,liver protection,anti-inflammatory,diuretic,anti-oxidation,anti-tumor and hypoglyce-mic activity;Because it is rich in nutrients such as vitamins,polysaccharides,amino acids,proteins,etc.,it has also been researched and developed into foods such as Hovenia dulcis vinegar,Hovenia dulcis wine,Hovenia dulcis beverage,etc.At present,the pharmacological activity of Hovenia dulcis has been extensively studied at domestic and abroad,but after literature research,no relevant reports about Hovenia dulcis and anti-calculus activity have been found.In this paper,Hovenia dulcis is the research object,and its extracts are obtained through in vivo and in vitro pharmacological experiments.anti-calculus activity and its preliminary mechanism are studied and evaluated.The main research work of this thesis is as follows:（1）In vitro cell experiment calcium oxalate monohydrate（COM）was used to stimulate human renal tubular epithelial cells（HK-2）for 24 h to construct an in vitro calcium oxalate kidney stone model.After successful modeling,potassium citrate was used as a positive control.Hovenia dulcis extract was used in high,medium and low doses to act on the calcium oxalate kidney stone model in vitro for 24 hours.The HK-2 cell model lactate dehydrogenase（LDH）,superoxide dismutase（SOD）,malondialdehyde（MDA）,reactive oxygen species（ROS）content changes,cell viability,and apoptosis indicators was tested.To evaluate the in vitro of hovenia dulcis extract Anti-calculus activity;The results of the study showed that in the cell experiment,compared with the model group,the content of SOD,LDH and cell viability of HK-2 cells in the normal group,potassium citrate group,and hovenia dulcis extract high-dose group increased,while the content of MDA and ROS decreased.There are extremely significant differences（P<0.01）.In the medium-dose group,SOD,LDH content and cell viability increased,and there were extremely significant differences（P<0.01）,MDA and ROS content decreased,with significant differences（P<0.05）.In the low-dose group,cell viability showed an upward trend,which was significantly different from the model group（P<0.05）;（2）Real time PCR and Western Blot methods were used to study OPN m RNA and KIM-1 m RNA,Osteopontin（OPN）and Kidney injury molecule-1（KIM-1）in HK-2 cells in each experimental group the amount of expression.The results of the study showed that:in the preliminary mechanism study of the cell experiment,compared with the model group,the expression of OPN m RNA and KIM-1 m RNA in the normal group decreased significantly,and the difference was extremely significant（P<0.01）,the expressions of OPN m RNA and KIM-1 m RNA in the potassium citrate group and the hovenia dulcis extract high-dose group were significantly decreased,with significant differences（P<0.05）;The expression of KIM-1 m RNA in the medium dose group of Hovenia dulcis extract decreased,and the difference was significant（P<0.05）.The expression of OPN m RNA decreased slightly,and the statistical analysis showed no significant difference（P>0.05）.The expression of OPN m RNA and KIM-1 m RNA in the low-dose Hovenia dulcis Thunb extract group showed a relative-ly small downward trend,and there was no significant difference（P>0.05）.Compared with the model group,the expression of OPN and KIM-1 protein in the normal group,potassium citrate group and Hovenia dulcis extract high dose group was significantly decreased,and the difference was extremely significant（P<0.01）.The expression of OPN protein in the middle dose group was relatively small,but there was also significant difference（P<0.05）.The expression of KIM-1 protein decreased significantly（P<0.01）.（3）In vivo animal experiments use ethylene glycol combined with ammonium chloride to construct an in vivo calcium oxalate kidney stone model;After successful modeling,After successful modeling,with Paishi granules as a positive control,the rats were given high,medium and low doses of Hovenia dulcis extract respectively for 2 weeks;After the administration,the urine of the rats on an empty stomach for 24h was collected,then its kidney weighed after anesthetizing and dissecting the rats,and the kidney coefficient calculated;Detect Ca²⁺in urine,Ca²⁺in kidney tissue homogenate,Ca²⁺in serum,Serum creatinine（SCR）,serum urea nitrogen（BUN）,uric acid（UA）content;make mineralization knots nodal staining（VK）section,observe the pathological changes of kidney tissue,calculate the percentage of expression area of mineralized nodules to evaluate the anti-calculus activity of hovenia dulcis extract in vivo;The results of the study show that in animal experiments,compared with the model group,the positive control group,the high-dose hovenia dulcis extract,the middle-dose group SCR,BUN,UA,urine Ca²⁺,serum Ca²⁺content,and mineralized nodule expression the area percentages were all decreased,and the difference was significant（P<0.05）;there was no significant difference in the urinary output of rats in each group after 24 h fasting（P>0.05）.In addition,there was no significant difference in indicators between the Paishi granule group and the Hovenia dulcis extract high-dose group（P>0.05）.（4）The anti-calculus mechanism of hovenia dulcis extract was mainly used western blot method to evaluate the expression of OPN and KIM-1 in the kidney tissue of experimental rats.The research results show that:compared with the model group,the expression of OPN and KIM-1 in the normal group,the positive control group,the hovenia dulcis extract,and the middle-dose group were significantly decreased,There is an extremely significant difference（P<0.01）.In the low-dose group,the downward trend of KIM-1 protein expression was relatively small,but there were also significant differences（P<0.05）.In summary,in the cell experiment,hovenia dulcis extract has a certain alleviating effect on the in vitro calcium oxalate kidney stone model established by COM-stimulated HK-2;at the same time,it has a certain effect on OPN m RNA and KIM-1 m RNA and OPN in HK-2 cells.,KIM-1 expression has a down-regulating effect.In animal experiments,Hovenia dulcis extract also has a certain improvement effect on the calcium oxalate kidney stone model induced by ethylene glycol and ammonium chloride,and also has a down-regulation effect on the expression of OPN and KIM-1 in rat kidney tissue.And among the indicators tested in this research,the anti-calculus activity of hovenia dulcis extract in the high-dose group was similar to that of the positive control group,even in SCR,BUN,urine Ca²⁺,renal tissue Ca²⁺,serum Ca²⁺,MDA among the indicators,the anti-calculus activity of hovenia dulcis extract high-dose group was slightly better than that of the positive control group.In this study,the anti-calculus activity of hovenia dulcis in vivo and in vitro and its preliminary mechanism were studied,laying the foundation for the application and development of the anti-calculus activity of hovenia dulcis.