Abstract:

Objective To discuss the therapeutic effect of Mishitong capsule on calculi-inducing solution-induced kidney stone mice，and to clarify its mechanism. Methods A total of 50 SPF-grade male ICR mice were selected. Forty mice were randomly selected and intragastrically administered with 3 mL·d^-1 calculi-inducing solution （0.75% ethylene glycol+0.75% ammonium chloride） for 2 weeks to establish the kidney stone mouse model， and the remaining 10 mice served as control group. After successful modeling， the 40 model mice were randomly divided into model group， low dose of Mishitong capsule group， high dose of Mishitong capsule group， and positive drug group， with 10 mice in each group. One week after modeling， the mice in low dose of Mishitong capsule group and high dose of Mishitong capsule group were intragastrically administered with 225 and 450 mg·kg^-1 Mishitong capsule suspension， respectively； the mice in positive drug group were intragastrically administered with 5 g·kg^-1 Paishi granule suspension； the mice in control group and model group were intragastrically administered with an equal volume of normal saline. The administration was performed for 2 consecutive weeks， during which the body mass of the mice was measured weekly. After administration， the mice in various groups were sacrificed， renal tissue was isolated， and serum and feces were collected. Kit method was used to detect the levels of reduced glutathione （GSH） and malondialdehyde （MDA） and the activity of superoxide dismutase （SOD） in renal tissue of the mice in various groups， as well as the levels of blood urea nitrogen （BUN）， creatinine （Cr）， calcium （Ca）， phosphorus （P）， and magnesium （Mg） in serum of the mice in various groups. Western blotting method was used to detect the expression levels of nuclear factor erythroid 2-related factor 2 （Nrf2）， autophagy-related protein 5 （ATG5）， P62， and heme oxygenase-1 （HO-1） proteins in renal tissue of the mice in various groups； 16S ribosomal RNA （rRNA） sequencing method was used to analyze the gut microbiota diversity of the mice in various groups. Results Compared with control group， the mice in model group showed a slight decrease in body mass in the early modeling stage， accompanied by mental lethargy and piloerection. Compared with model group， the declining trend of body mass was slowed， and the mental status was improved in the mice in low dose of Mishitong capsule group and high dose of Mishitong capsule group. The kit method results showed that compared with model group， the GSH levels and SOD activities in the renal tissue of the mice in low dose of Mishitong capsule group， high dose of Mishitong capsule group， and positive drug group were significantly increased （P<0.05）， and the MDA levels were significantly decreased （P<0.05）. Compared with model group， the BUN and Ca levels in the serum of the mice in low dose of Mishitong capsule group and high dose of Mishitong capsule group were significantly decreased （P<0.05 or P<0.01）， and the Mg level was significantly increased （P<0.05）； the Cr and P levels in the serum of the mice in high dose of Mishitong capsule group were significantly increased （P<0.05）； the BUN， Cr， Ca， and P levels in the serum of the mice in positive drug group were significantly decreased （P<0.05 or P<0.01）， and the Mg level was significantly increased （P<0.05）. The Western blotting results showed that compared with model group， the expression levels of HO-1， Nrf2， and ATG5 proteins in the renal tissue of the mice in low dose of Mishitong capsule group， high dose of Mishitong capsule group， and positive drug group were significantly increased （P<0.05）， and the expression levels of P62 protein were significantly decreased （P<0.05）. The 16S rRNA sequencing results showed that compared with model group， the Chao1 index and Pielou’s evenness index of gut microbiota of the mice in high dose of Mishitong capsule group and positive drug group were significantly increased （P<0.05）. At the phylum level， compared with model group， the relative abundance of Bacteroidetes in gut microbiota of the mice in low dose of Mishitong capsule group and high dose of Mishitong capsule group was decreased， and the relative abundances of Firmicutes and Proteobacteria were increased； at the genus level， compared with model group， the relative abundance of Prevotella in gut microbiota of the mice in high dose of Mishitong capsule group was increased， the relative abundance of Bacteroides was decreased， and a new dominant genus Helicobacter emerged. Conclusion Mishitong capsule ameliorates the pathophysiological damage in calculi-inducing solution-induced kidney stone mice， and its mechanism is potentially associated with reducing serum BUN and Cr levels， modulating serum Ca， P， and Mg levels， alleviating oxidative stress injury， regulating the expression of proteins related to the Nrf2/ATG5/P62 signaling pathway， and improving the gut microbiota structure.

Key words: Renal calculi, Mishitong capsule, Oxidative stress, Nuclear factor erythroid 2-related factor 2 signaling pathway, Autophagy, Intestinal flora