Renal-Protective Effects and Potential Mechanisms of Traditional Chinese Medicine after Ischemia-Reperfusion Injury

Renal ischemia-reperfusion (I/R) injury mainly causes acute kidney injury (AKI) after renal transplantation, trauma, sepsis, and hypovolemic shock. Patients with renal I/R injury are frequently associated with a poor prognosis. Traditional Chinese medicine (TCM) has been used for the prevention and treatment of various diseases in China and other Asian countries for centuries. Many studies have shown the protective effect of TCM on renal I/R injury, due to its diverse bioactive components. The potential mechanisms of TCMs on renal I/R injury include anti-inflammation, antioxidative effect, anti-cell death, downregulation of adhesion molecule expression, regulation of energy metabolism by restoring Na+-K+-ATPase activity, and mitochondrial fission. This review summarizes the major developments in the effects and underlying mechanisms of TCMs on the renal I/R injury.


Introduction
Renal ischemia-reperfusion (I/R) injury is the main cause of acute kidney injury (AKI) and is reported to be associated with high morbidity and mortality in both adults and children. It often occurs after renal transplantation, as well as in sepsis, trauma, hypovolemic shock, etc. [1]. During the renal I/R process, excess reactive oxygen species (ROS) are likely to cause oxidative stress, which subsequently triggers lipid peroxidation [2] and cell death due to apoptosis, autophagy, pyroptosis, and ferroptosis caused by DNA and protein damage in ischemic tissue [3]. In addition, inflammation plays an important role in renal I/R injury, along with leukocyte infiltration and tissue damage in the kidney, followed by excessive production of proinflammatory cytokines [4]. Energy metabolism dysfunction, such as decreased Na + -K + -ATPase activity, has been considered as one of the critical mechanisms of renal I/R injury [5].
Increase in blood urea nitrogen (BUN) and creatinine (Cr) [3] and a higher urinary total protein level are reported to occur after renal I/R injury [6]. Histopathology of renal I/ R injury shows luminal narrowing, intraluminal necrotic cellular debris, tubular basal membrane rupture, tubular vacuolization, interstitial congestion, apparent cell swelling and nuclear infiltration in the H&E staining of renal tissues [7], and higher Kim-1 expression, thus causing a higher renal tubular injury score [2].
Traditional Chinese medicine (TCM) has been used in China, Korea, Japan, and other Asian countries for the treatment of various diseases, such as cerebrovascular diseases, cardiac diseases, liver diseases, renal disorders, etc. In the theory of TCM, renal I/R injury is attributed to Qi and blood deficiency and blood stasis syndrome [8]. From the perspective of TCM, renal I/R injury should be treated through the replenishment of Qi and activation of blood circulation [7]. Several TCMs have been applied for the prevention and treatment of renal I/R, and they show different renal-protective effects in Table 1, including antiinflammation (Cordyceps sinensis, cordycepin, Yisheng injection, cryptotanshinone, Panax notoginseng, total glucosides of paeony, hydroxysafflor yellow A, farnesiferol B, anemoside B4, etc.), antioxidative stress (ligustrazine, berberine, glycyrrhizin, Sheng Mai San, hyperoside, salvianolic acid A, salvianolic acid B, arctigenin, farnesiferol B, polydatin, Shenfu injection, etc.), anti-cell death (honokiol, cryptotanshinone, brazilin, salvianolic acid B, Sanqi oral solution, etc.), downregulation of adhesion molecule expression (ligustrazine and Yisheng injection), regulation of energy metabolism by restoring Na + -K + -ATPase activity (Fufang Shenhua Tablet), and mitochondrial fission (hyperoside).
In this review, we have discussed the studies in the past decades on the protective effects and potential mechanisms of TCMs and the major ingredients on renal I/R injury.

Protective Effects of TCMs and Major Ingredients on Renal I/R Injury
Generally speaking, almost all the following TCMs and the major ingredients play a protective role in renal I/R injury, including the improvement of renal function and histological changes in renal tissues, inhibiting inflammatory cytokine release and macrophage/neutrophil infiltration, decreasing production of oxidative stress and lipid oxidation, regulating programmed cell death (apoptosis, autophagy, pyroptosis, or ferroptosis), decreasing release of adhesion molecules, regulating energy metabolism, endothelial injury, and mitochondrial dysfunction [25].

Renal Function and Renal Histological Examination.
In previous studies, renal function was often measured by the levels of Scr and BUN. In some cases, urinary total protein level was also used for renal function measurement. It was reported that Scr and BUN levels were significantly increased in the I/R model group, compared with the sham group. TCMs or CCMs, such as Fufang Shenhua Tablet, cryptotanshinone, and notoginsenoside R1, could inhibit the increased levels of Scr and BUN, and thus improve renal function [13,17,19]. In addition, anemoside B4 could decrease the urinary total protein level [6], indicating improvement of renal function. Several histological abnormalities were observed after renal I/R injury, including tubular brush border loss, epithelial cell dilatation and necrosis, cytoplasmic  [3]. Most TCMs were reported to alleviate renal histological changes and decrease renal tubular injury score, caused by I/R injury. Furthermore, TCMs, such as Sanqi oral solution, hydroxysafflor yellow A, and total glucosides of paeony, showed dosedependent effects, such that higher concentrations showed greater alleviating effects [4,7,23]. In contrast, ATG played a detrimental role in renal I/R injury since renal function and histology worsened with increasing concentration [3].

Antioxidative Stress.
Oxidative stress and lipid oxidation increase after renal I/R injury, while treatment with some TCMs inhibits the production of oxidative stress and promotes the production of antioxidant factors. In Feng Han and AR Shahed's studies [10,26], Cordyceps sinensis and its extract cordycepin increased the levels of antioxidative factors, including GSH, GSHPx, and SOD, and decreased the levels of oxidative stress products, including MDA, NO, and iNOS. Other TCMs also showed similar effects, such as MDA decrease and SOD increase in the ligustrazine treatment group [11], berberine treatment group, and Sal A and Sal B treatment groups [21,22], SOA decrease and SOD increase after glycyrrhizin treatment [15], MDA/MPO decrease and SOD/CAT increase after honokiol treatment [16], decrease in iNOS and ONOOlevels after SMS treatment [27], and decrease in ROS levels after hyperoside treatment. CS treatment decreased NGAL levels both in kidney tissues and in urine, which was increased in the I/R group [28]. In addition, farnesiferol B reduced oxidative stress (diminished levels of NAGL and H2O2) and lipid oxidative signaling pathways (diminished levels of lipid peroxidation markers, including 4-HNE and MDA) in renal I/R [2].

Anti-Cell Death.
ere are several types of programmed cell death, including apoptosis, autophagy, pyroptosis, and ferroptosis. Previous studies have reported that the protective effect of TCMs on renal I/R injury was associated with programmed cell death, especially apoptosis.
Antiapoptosis was observed after treatment with some TCMs before or after renal I/R injury, with downregulation of the levels of apoptosis proteins and upregulation of the levels of antiapoptosis proteins. In the Cordyceps sinensis treatment group [26], Fas and FasL mRNA expression levels were decreased, while caspase-3 activity was decreased after glycyrrhizin and honokiol treatment [15,16]. TUNELpositive cells were increased after Sal A treatment, indicating a reduction of apoptotic activity [21]. An in vitro study found that berberine significantly inhibited the expression of apoptotic proteins, such as Bax [14].
Treatment with Sanqi (SQ) oral solution showed decrease in apoptosis and increase in autophagy [7]. Lower Bax and caspase-3 levels and higher Bcl-2 level were observed in the SQ treatment group in a dose-dependent manner, demonstrating the antiapoptotic activity of SQ. Meanwhile, compared to the I/R model group, higher dose of SQ significantly increased the levels of LC3II/LC3I and Beclin1, indicating an enhanced autophagy activity after renal I/R injury. In addition, treatment with SQ and 3-MA, an autophagy inhibitor, showed worse histological damages and renal function, compared with SQ alone treatment, indicating the essential role of autophagy in SQ treatment during renal I/R injury.
In Yu Pang's study [22], the levels of GSDMD, caspase-1, and IL-1β, the pyroptosis-related proteins, were strongly upregulated in the model group.
e pyroptosis-related proteins were significantly decreased after treatment with Sal B, demonstrating the antipyroptosis activity of Sal B.
Lastly, ferroptosis plays an important role in the protective effects of farnesiferol B treatment on renal I/R injury. e mRNA expression of Gpx4, the key ferroptosis regulator, was significantly reduced after renal I/R injury, while farnesiferol B treatment increased its expression. However, the intergroup difference was not significant [2]. So, further studies are needed.

Downregulation of Adhesion Molecules.
In the I/R model group, expression of ICAM-1 was upregulated but was greatly diminished after ligustrazine treatment [11]. Moreover, ICAM-1 was downregulated in a dose-dependent manner after pretreatment with Yisheng injection in the renal I/R group [12].
2.6. Recovery of Na + -K + -ATPase Activity. Yang Yang et al. [5] found a significant decrease in Na + -K + -ATPase activity in the I/R model group when compared with the sham group. However, Shenhua Tablet (SHT) was observed to increase the decreased Na + -K + -ATPase activity after renal I/ R, both in the low-dose group and the high-dose group. Combined with other results in this study, the authors concluded that SHT could recover the number of Na + -K + -ATPase and improve its activity in the tubular epithelial cells, promote epithelial cell polarity in renal tubular cells, and thereby improve renal function.

Endothelial Protective Effects. Salvianolic acid A (SAA)
can protect peritubular capillary endothelium from renal I/R injury [29]. VEGFA expression was increased in tubular epithelial cells, which was associated with peritubular capillary density in the SAA treatment group, as well as the reduced levels of plasma vWF and lower platelet activation after I/R injury. Moreover, significant increase in Klotho protein expression was observed, indicating less endothelial injury after renal I/R with SAA treatment.

NF-κB Signaling
Pathway. NF-κB is believed to play an important role in the anti-inflammatory effects and antiapoptotic and antioxidative activity of TCMs in renal I/R process. Activation of the NF-κB signaling pathway was inhibited in the cryptotanshinone (CTS) treatment group after renal I/ R [17]. I/R significantly increased the phosphorylated p65 and IκBα levels, indicating the activation of NF-κB signaling pathway. After CTS treatment, the phosphorylation of p65 and IκBα was inhibited. Brazilin was also proven to downregulate the inflammatory activity by inhibiting NF-κB signaling pathway activation in vitro [20].
Hydroxysafflor yellow A (HSYA) was reported to decrease the renal inflammation after I/R injury by suppressing the TLR4/NF-κB signaling pathway activation [4], as phospho-IKKβ, phospho-IκBα, and NF-κB-p65 expression levels were upregulated in the I/R group but were downregulated after treatment with HSYA in vitro. Similar effect of arctigenin (ATG) and polydatin on TLR4 and NF-κB-p65 expression was observed [3,31].
Notoginsenoside R1 (NR1) decreased inflammatory factors and apoptosis by inhibiting the phosphorylation of p38 MAPK and NF-κB activation in renal tissue after I/R injury [12]. e anti-inflammatory effects and antioxidative activities of farnesiferol B on renal I/R injury were based on the activation of TGR5 and inhibition of the NF-κB pathway [2].

Nrf2
Signaling Pathway. In renal I/R process, Nrf2 plays a central role in renal-protective effects of TCMs, including the anti-inflammatory effects and antioxidative and anti-cell death activity.
Cordycepin is known to alleviate inflammation, oxidative stress, and apoptosis in the I/R process. e antioxidative mechanism of cordycepin was analyzed by measuring the levels of Nrf2 and HO-1, the key proteins in antioxidative stress process. Western blotting results showed an increase in the expression of Nrf2 and HO-1 after cordycepin treatment, indicating that cordycepin may suppress the oxidative stress during renal I/R injury through the Nrf2/ HO-1 signaling pathway [10].
Another study found that Sal B could inhibit caspase-1/ GSDMD-mediated pyroptosis and thereby alleviate I/R injury in mice, by activating Nrf2/NLRP3 signaling pathway [22]. Sal B was reported to inhibit pyroptosis by downregulating pyroptosis-related proteins GSDMD, caspase-1, and IL-1β. Moreover, Sal B showed its antioxidative property by inhibiting the expression of Keap1 protein, thus increasing Nrf2 nuclear expression and HO-1 protein levels in vivo. In vitro, Sal B reduced NLRP3 protein expression and increased Nrf2 nuclear import and activated expression of downstream antioxidant components. Moreover, the Nrf2/ NLRP3 signaling pathway was demonstrated to be involved through Nrf2 knockdown in vivo and siNrf2 transfection in vitro.

Sonic Hedgehog (Shh) Signaling Pathway.
Shh signaling pathway is important in the antioxidative and antiapoptotic effects of TCM treatment after renal I/R injury. Polydatin alleviated apoptosis and oxidative stress after renal I/R injury through activating the sonic hedgehog signaling pathway [24]. Polydatin was found to dose-dependently induce increase in the Shh gene and protein expression compared with the I/R group, both in vitro and in vivo. Moreover, the mRNA and protein expression of Ptch 1 and Smo, two key elements in the Shh signaling pathway, were reduced by cyclopamine (an inhibitor of Smo) combined with polydatin. Meanwhile, higher apoptotic activity and oxidative effects were observed. erefore, inhibition of Shh signaling impaired the antioxidative and antiapoptotic effects of polydatin.

Summary
is review discusses the protective effects of TCMs and their active components on renal I/R injury. e mechanisms of the protective effects of TCMs include inhibition of inflammation, decrease of oxidative stress and lipid oxidation products, regulation of programmed cell death, reduction of adhesion molecule release, and regulation of energy metabolism and endothelial injury by activating NF-κB and Nrf2 signaling pathways, which are considered to be multiple targets for successful treatment. However, the existing studies are either animal experiments in vivo or in vitro studies, with lower level of evidence and strength of recommendation.
erefore, further clinical studies are needed to explore the effects of TCMs and the underlying mechanisms.

Conflicts of Interest
e authors declare that they have no conflicts of interest. 4 Evidence-Based Complementary and Alternative Medicine