Diuretic activity and mechanism of Mosla chinensis Maxim. cv. Jiangxiangru in normal Rats

Background: Jiangxiangru, a commonly used clinical Chinese medicine, has been investigated mainly over its effects on Summer heatstroke and the dampness inside the spleen and stomach. This study focuses on the diuretic effect of Jiangxiangru in normal rats as well as the underlying mechanism. Methods: 48 rats were randomly divided into the model group (saline), the positive control group (furosemide 0.02g/kg), low and high dosage of water decoction of Jiangxiangru group (0.2 g/ml and 1g/ml), low and high dosage of water extract of Jiangxiangru group (0.1463g/ml and 0.7317g/ml), and the low and high dosage of volatile oil of Jiangxiangru group (0.1791g/ml and 1.4328g/ml). Each group was assigned 6 rats. Thirty minutes after the successful establishment of the model, the rats were treated orally with corresponding doses of drugs at 10 ml/kg. The control group received an equivalent amount of water. After administration, Urine volume at each time point was measured and urine K + , Na + , Cl content were measured. The contents of ALD, ANP, ADH, Na + -K + -ATPase, the gene expression and protein levels of AQP 1 , AQP 2 and AQP 3 in urine of rats in each group were determined. SPSS22.0 statistical software was used to analyze the experimental data. One way ANOVA and multiple comparisons between LSD groups were performed. The difference is considered signicant When p < 0.05 or p < 0.01. Results: Our results showed that Jiangxiangru decoction group has the strongest diuretic effect, followed by water extract, then the volatile oil. The diuretic mechanism of Jiangxiangru may affect the kidney in addition to the hormone level related to the change of body uid metabolism. The distal convoluted tubules are related to the content of aquaporin AQP in the collecting tube, thereby inhibiting water reabsorption. Conclusion: The contents of Na + , K + , and Cl - and PH in urine of rats don’t change signicantly in this experiment. The


Background
Diuretics have wide clinical application in treating edema caused by various reasons. They can also be used to treat hypertension(Xing hai ning, Li chen et al. 2016), kidney injury (Jeon, Kim et al. 2018), chronic heart failure (Qavi, Kamal et al. 2015), and other diseases (Alhanafy, Labeeb et al. 2018). As the medical research towards diuretics deepened, the increase in observed post-treatment clinical adverse reactions (Yang 2018) has turned the public attention to traditional Chinese medicines (TCMs) (Lin, Pan et al. 2014, Liu, Yang et al. 2019. In China, several TCMs are used as diuretic drugs (Jia and Jia 2016, Li, Cai et al. 2016, Gao, Bai et al. 2018. However, most of them are lacking pharmacological studies to reveal the molecular pathways that might be contributing to diuretic effects. Mosla chinensis Maxim. cv. Jiangxiangru (Chinese: Jiangxiangru, ) is one of these TCMs. It belongs to the the Labiates family and is a famous herbal TCM that is described in the authoritative medical book of Chinese Pharmacopoeia (CHINA 2015).
Elsholtzia is the aerial part of Mosla chinensis Maximak or M. chinensis Jiangxiangru (Liu, Wang et al. , Zhimin, Wang et al. 2016). Jiangxiangru, a herb mainly distributed in Yichun and Xinyu in Jiangxi Province of China, is genuinely a regional herb used in the Traditional Chinese Medicine (Liu, Wang et al. , Qi, Fang et al. 2015, Zhimin, Wang et al. 2016) (Chinese Pharmacopoeia). Elsholtzia can relieve the sweating caused by the heat in summer, and the effect is like ephedrine in winter (XianGuo 2013(XianGuo , lingyuan 2015. It tastes mild and warm, with effects including perspiration, diuresis, and resolving swelling, and dehumidi cation. Modern research shows that there are many chemical components in Jiangxiangru, such as avonoids, terpenoids, glycosides, steroids, coumarins, organic acids, fats, and lignans (Hu, Xie et al. 2010). Different extracts of Jiangxiangru contain different active ingredients, and their pharmacological effects may also be different. Therefore, to increase the e ciency of further development of Jiangxiangru product and their usage, we tested the diuretic effects of Jiangxiangru over the decoction group, the water extract and the volatile oil. Amongst them, volatile oil is the main active ingredient and has antipyretic, anti-in ammatory and analgesic effects (Luo, Yang et al. 2006, Jiang, Lu et al. 2007, Hu, Xie et al. 2010, Li, Cui et al. 2014, Zhimin, Wang et al. 2016. The purpose of the study was to evaluate the diuretic activities in normal rats. We assessed the urine output volume, the urinary electrolyte concentrations (Na + , K + and Cl -) and pH value, the levels of Na + -K + -ATPase, atriopeptin (ANP), anti-diuretic hormone (ADH) and aldosterone (ALD), as well as AQPs levels for e cacy evaluation.

Methods study chemicals and reagents
Jiangxiangru (batch number: 180823) was purchased from Jiangxi Jiangzhong Traditional Chinese Medicine Decoction Co., Ltd. The Jiangxiangru was placed in a round-bottom ask. After soaking in the proportion of distilled water to 1:10 for 30 minutes, the volatile oil was extracted by steam distillation for 5 hours then collected (yield: 0.67%). The volatile oil was prepared into 200 ml low and high concentration volatile oil solution with distilled water containing 2% Tween-80, respectively. The reduced crude drug amounts were 0.1791 and 1.4328 g/ ml, respectively. The water extract with low and high concentration was obtained by condensed extractpart of the extract, and the equivalent crude drug dosage was 0.1463 and 0.7317g/ml, respectively. Jiangxiangru decoction is decocted by pharmacy The 50 rats were isolated for one week. After the isolation rats were placed in metabolic cage for 3 days; then on the 4 th day, fasting (no water) for 18 hours. The rats were given 2.5ml/100g saline water to record the urine volume for 2 hours. The rats whose urine volume was more than 40% of the water supply were successfully established as saline water load model (Chen, Feng et al. , Feng, Chen et al. , Gao, Bai et al. 2018). It can be used in the following experiments.
48 rats were randomly divided into 8 groups: the model group (saline), the positive control group (furosemide 0.02g/kg), low and high dosage of water decoction of Jiangxiangru group (0.2 g/ml and 1g/ml), low and high dosage of water extract of Jiangxiangru group (0.1463g/ml and 0.7317g/ml), and the low and high dosage of volatile oil of Jiangxiangru group (0.1791g/ml and 1.4328g/ml). Each group was assigned 6 rats. According to clinical TCM practice the dosage of Jiangxiangru for adults (60 kg/person) is 10~30 g/kg/day (Editorial Committee of Zhonghua Bencao National Traditional Chinese Herb Administration, 1999). For rats, this dosage is 0.166~1.5g/kg/day (raw herb). Thirty minutes after the successful establishment of the model, the rats were treated orally with corresponding doses of drugs at 10 ml/kg. The control group received an equivalent amount of water.
After administration, rats in each group were immediately put into metabolic cages. Urine samples were collected after 1,2,3,4,5,6,24,48,72,96,144 and 168 hours respectively. Urine volume at each time point was measured and urine K + , Na + , Clcontent were measured with Beckman AU2700 biochemical analyzer. At the end of the experimental period, 4 mL of blood were collected in vacuum tubes from the abdominal aorta, allowed to clot on ice, and subsequently subjected to centrifugation at 3000 rpm at 4°C for 10 min. The supernatant was stored at -80°C for further analysis. All of the rats were sacri ced by cervical dislocation. The contents of ALD, ANP, ADH, Na + -K + -ATPase, the gene expression and protein levels of AQP 1 , AQP 2 and AQP 3 in urine of rats in each group were determined.

Biochemical methods
Enzyme-linked immunosorbent assay (ELISA) kit was used to analyze ALD, ANP, ADH, Na + -K + -ATPase, AQP 1 , AQP 2 and AQP 3 in urine. Rat urine was centrifuged at 3000 rpm for 10 min at 4 ℃ in an Eppendorf centrifuge. The supernatant was collected, and recombinant rat Na + -K + -ATPase, ANP, ADH, ALD, AQP 1 , AQP 2 and AQP 3 were used to construct standard curves. The absorbance of standards and samples was determined spectrophotometrically at 450 nm using a Multiskan GO (Thermo scienti c, USA) microplate reader. The results were plotted against the linear portion of a standard curve.

RNA-isolation and qPCR
Renal medulla samples were cleaved by Trans Zol Up homogenized and added chloroform. The solution was divided into colorless aqueous phase and pink organic phase. RNA was adsorbed in aqueous phase by silica gel membrane centrifugal column. RNA was prepared by reverse transcription using a RNA kit.
Real-time PCR was performed using Power SYBR Green PCR Master Mix and Corbbet Cycler. Each reaction was performed in 40 cycles with a total volume of 10 μl.
Primer sequences and PCR ampli cation reactions were as according to the literature (Yan, Zeng et al. 2014). The internal reference gene of the experiment was GAPDH (Zhong, Li et al. 2018). The primer sequences and reaction systems are shown in Tables 1 and 2. The mixed test solution was placed in a uorescence quantitative PCR machine for reaction. The ampli cation reaction conditions were as following: 95 ° C for 30 s; 95 ° C for 10 s, 60 ° C for 1 min, 40 cycles; then the reaction temperature was raised from 50 ° C to 90 ° C (1 ° C every 6 s); The ct values of the reference gene and the target gene were collected; nally, the relative expression level of the target gene was calculated, and the data was processed according to the experimental method of Yang et al (Yang, Liu et al. 2015) Statistics analysis SPSS22.0 statistical software was used to analyze the experimental data. Data were expressed as mean ± standard deviation (x ±s). One way ANOVA and multiple comparisons between LSD groups were performed. The difference is considered signi cant When p < 0.05 or p < 0.01.

Effect on urinary excretion volume
In order to determine whether Jiangxiangru has diuretic effect, we measured the urine volume of rats treated with Jiangxiangru or furosemide at 1,2,3,4,5,6,24,48,72,96,120,144 hours. We found that 1 hour after administration, the urine volume of the low and high dosage of decoction of Jiangxiangru group, the high dose of volatile oil of Jiangxiangru group and positive control group increased signi cantly compared with the model group (P < 0.01 or P < 0.05). The effect of the high dose of decoction of Jiangxiangru group was the most obvious, and the urine volume of other groups also increased, there was no signi cant difference (P > 0.05). At 2 hours after administration, the urine volume of rats in positive control group increased, but there was no signi cant difference (P > 0.05). At 3-5 hours after administration, the urine volume of rats in each group had no signi cant difference (P > 0.05); but at 24 hours and 96 hours after administration, the urine volume of rats in high-dose of decoction of Jiangxiangru group increased, the difference was statistically signi cant(P < 0.05). This indicated that the water decoction of Jiangxiangru had a longer effect than furosemide.
From Figure 1, compared with model group, the total urine volume of rats in high dose of decoction of Jiangxiangru group and positive control group increased at 1h,6h,96h,120h,144h hours with statistical signi cance (P < 0.05), indicating that the water decoction of Mosla chinensis has obvious diuretic effect.

Effect on urinary pH and electrolyte concentrations
The changes of urinary ph and electrolytes in urine of rats at 1, 2, 3, 4, 5, 6, 24, 48, 72, 96, 120, 144 hours were observed (Fig. 2, Fig. 3). Compared with the model group, the contents of Na + , K + , and Clin the urine of rats in the administration group did not change signi cantly, and the pH of the urine of rats in each group had no signi cant difference (P > 0.05).
Effect on urine levels of Na + -K + -ATPase, ALD, ANP, ADH ELISA kit was used to verify whether Jiangxiangru could affect the contents of ALD, ANP, ADH and Na + - furosemide of AQP 1 level signi cantly lower than that of the control group (P < 0.01). Low dosage of decoction group (0.2g/ml), low dosage of water extract group(0.1463g/ml), and high dosage of volatile oil group had signi cant difference in AQP 2 (P < 0.01). The AQP3 protein content of low-dose group of Jiangxiangru decoction, volatile oil group, and furosemide group were signi cantly lower than that of model control group (P < 0.01).

Discussion
Urine is the most intuitive indicator to detect whether drugs have diuretic effect. Urine production includes three processes: glomerular ltration, renal tubule and collecting duct reabsorption and secretion. Diuretics are medications producing diuretic effects of varying strength by acting on different parts of the renal tubule, thus affecting the pathway of urine production. The excretion of electrolytes is also related to the site of drug action. Classic diuretics mainly exert diuretic effect by increasing the excretion of Na + and Clafter drug intervention as Na + and Clexcretion positively in uences diuresis. For example, furosemide mainly acts on the thick segment of the ascending branch of the medullary ridge, and reduces the reabsorption of Na + and Clby inhibiting Na + -K + -2Clco-transport, increases the excretion of NaCl, and increases the urinary H + and K + Excretion, which can cause hypokalemia. Therefore, it is crucial to determine the contents of Na + , K + , and Clin urine of rats treated with Jiangxiangru. Jiangxiangru and its 3 delivery reagents had no effects on contents of Na + , K + , and Clin urine. Therefore, we believe that the diuretic effect of Jiangxiang and each of it treatment group is not achieved by affecting Na + -K + -2Clco-transport.
The main function of ALD synthesized and secreted by adrenocortical globular zone cells is to increase the re-absorption of Na + and water, promote K + excretion and maintain the balance of water-salt metabolism. The active polypeptide ANP synthesized and secreted by the atrium is also known as atrial natriuretic factor or atrial natriuretic peptide. Its main functions are natriuretic, diuretic, inhibiting rennin release and ALD secretion. ADH is secreted by the hypothalamus and its main function is to promote water reabsorption. The diuretic activity and mechanism of drugs are not only related to the levels of related diuretic hormones (ALD, ADH, ANP), but also to the levels of aquaporin in urine regulated by hormones. Therefore, this study explored the diuretic activity and its mechanism of Jiangxiangru related diuretic hormones (ALD, ADH, and ANP). Jiangxiangru and its 3 delivery reagents had no effects on the anti-metabolite enzymes. The results showed that the high dosage of decoction of Jiangxiangru group can increase the secretion of ALD and increase the secretion of ANP, especially the effect of ALD on doseresponse relationship, suggesting that the aqueous extract of Jiangxiangru has diuretic effect. There was also anti-diuretic effect, which is related to the multi-component and multi-target characteristics of traditional Chinese medicine. The volatile oil of Jiangxiangru also has similar action characteristics.
Additionally, from the in uence of gene expression and protein level of AQP, Jiangxiangru showed complex urinary production effects. The three preparations groups signi cantly down-regulated the expression of AQP 1 gene, and the protein level was signi cantly lower than that of the control group. The group of decoction showed an increase in the expression of AQP 2 and AQP 3 genes, and the protein also increased. The other groups down-regulated the expression of AQP 2 and AQP 3 genes, and the protein was lower than the control group.

Conclusion
The contents of Na + , K + , and Cland PH in urine of rats don't change signi cantly in this experiment. The diuretic effect of Jiangxiangru could not cause electrolyte disturbance and other adverse reactions. This fully re ects the complex composition of traditional Chinese medicine, the role of multi-target characteristics.

Declarations
Ethics approval and consent to participate All experiments were carried out in adherence with the guidelines of the Institutional Animal Care and Use Committee of China and were approved by the Animal Care and Research Committee of Jiangxi University of Traditional Chinese Medicine.
Consent for publication The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All participants agreed to publish.
Availability of data and materials The data used to support the ndings of this study are available from the corresponding author upon request.
Competing interests The authors have no con icts of interest to declare.  Figure 1 The volume of excreted urine was measured at 1,2,3,4,5,6,24,48,72,96,120,144 hours; cumulative values were reported as mean ± SD for 6 rats in each group. *P< 0.05 and **P< 0.01 compared to the controls group.

Figure 4
Effects of Jiangxiangru and its three groups together with furosemide on the levels of Na+-K+-ATPase from rat urine (mol/l) at 144 h after oral administration; the values were reported as mean ± SD for 6 rats in each group. *P< 0.05 and **P< 0.01 compared to the controls group.

Figure 5
Effects of Jiangxiangru and its three groups together with furosemide on the levels of ANP, ADH and ALD from rat urine (pg/mL) at at 144h after oral administration; the values were reported as mean ± SD for 6 rats in each group. *P< 0.05 and **P< 0.01 compared to the controls groups.

Figure 6
Effects of Jiangxiangru and its three groups together with furosemide on the levels of AQPs from rat urine and serum at 144h after oral administration; the values were reported as mean ± SD for 6 rats in each group. *P< 0.05 and **P< 0.01 compared to the controls groups. (6A: The changes of AQP1, AQP2 and AQP3 aquaporin in rat serum, 6B: the protein levels of urine AQPs)